Methods of Treating Neurological Disorders

ABSTRACT

The present invention provides methods of treating epilepsy and other neurological disorders. The methods generally involve administering to an individual in need thereof an effective amount of an agent that blocks a transforming growth factor-beta pathway.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/120,218, filed Dec. 5, 2008, which application isincorporated herein by reference in its entirety.

BACKGROUND

Epilepsy, affecting 0.5-2% of the population worldwide, is one of themost common neurological disorders. While the characteristic electricalactivity in the epileptic cortex has been extensively studied, themechanisms underlying epileptogenesis are poorly understood. Focalneocortical epilepsy often develops following traumatic, ischemic orinfectious brain injury. Under these conditions, local compromise ofblood-brain barrier (BBB) integrity is common, as revealed byultrastructural studies of animal and human epileptic tissue in multipleforms of epilepsy, raising the possibility that primary vascular damage,and specifically BBB opening, may serve as an initial event leading toepilepsy. This hypothesis has been confirmed by animal studies, in whichopening of the BBB was sufficient to induce delayed epileptiformactivity. Subsequent studies have shown that albumin, the most commonserum protein, is sufficient to recapitulate the epileptiform activityinduced by BBB disruption, and that albumin is selectively taken up byastrocytes (Ivens et al., 2007).

LITERATURE

-   Ivens et al. (2007) Brain 130:535-547; WO 2008/006583; WO    2008/066626.

SUMMARY OF THE INVENTION

The present invention provides methods of preventing and treatingepilepsy and other neurological disorders. The methods generally involveadministering to an individual in need thereof an effective amount of anagent that blocks a transforming growth factor-beta pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E depict the effect of serum albumin on epileptiform activityand TGF-β pathway activation.

FIGS. 2A-C depict genome wide transcriptional analysis followingepileptogenic treatments.

FIGS. 3A-F depict gene ontology annotation analysis.

FIG. 4A-F depicts qRT-PCR gene expression analysis.

FIG. 5 depicts the effect of albumin on TGF-β pathway gene expression.

FIGS. 6A-E depict the effect of blocking TGF-β signaling onalbumin-induced gene expression and epileptiform activity.

FIG. 7 depicts Western blot detection of phosphorylated Smad2 and Smad3proteins in sham-treated (a), BSA-treated (b), and BSA+losartan treatedrats.

FIG. 8 depicts changes in mRNA expression levels in animals treated withBSA or BSA and losartan for 24 hours.

FIGS. 9A-D depict EEG changes during epileptogenesis.

FIGS. 10 a-d depict transcriptional changes in astrocytes followingexposure to albumin or BBB disruption.

FIGS. 11 a-d depict alterations in astrocytic potassium and glutamateregulating genes.

FIGS. 12 a-e depict electrophysiological evidence for reduced glutamateand potassium buffering during epileptogenesis.

FIGS. 13 a-h illustrate application of NEURON-based model to determinethe effects of [K+]o accumulation.

FIGS. 14 a-d illustrate application of NEURON-based model to determinethe effects of glutamate accumulation.

FIGS. 15 a-d illustrate modelling effect of reduced potassium andglutamate clearance.

FIG. 16 a-d depict in vitro recording showing frequency-dependentincreased neuronal excitability and hyper-synchronous network activityduring albumin-mediated epileptogenesis.

DEFINITIONS

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to a mammal, including, but not limited to, murines,simians, humans, mammalian farm animals, mammalian sport animals, andmammalian pets.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aTGF-β receptor antagonist” includes a plurality of such antagonists andreference to “the active agent” includes reference to one or more activeagents and equivalents thereof known to those skilled in the art, and soforth. It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present invention provides methods of treating epilepsy and otherneurological disorders. The methods generally involve administering toan individual in need thereof an effective amount of an agent thatblocks a transforming growth factor-beta (TGF-β) pathway.

In some embodiments, an “effective amount” of an agent that blocks aTGF-β pathway is an amount that is effective to reduce the incidence ofan epileptic seizure in an individual by at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, or more than 50%, compared tothe incidence of epileptic seizure in the individual in the absence oftreatment with the agent.

In some embodiments, an “effective amount” of an agent that blocks aTGF-β pathway is an amount that is effective to reduce the durationand/or severity of an epileptic seizure in an individual by at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, or more than 80%,compared to the duration and/or severity in the individual in theabsence of treatment with the agent.

In some embodiments, an “effective amount” of an agent that blocks aTGF-β pathway is an amount that is effective to return an astrocyte froman activated state to a resting state. In some embodiments, an“effective amount” of an agent that blocks a TGF-β pathway is an amountthat is effective to reduce astrocyte dysfunction by at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or more than 80%, compared tothe astrocyte dysfunction in the absence of the agent. Astrocytefunction can be assessed using electrophysiological assays (e.g.,current recordings, voltage clamp recordings), assays designed to testextracellular K⁺ concentrations (e.g., using electrodes that measureextracellular K⁺ levels), and the like.

In some embodiments, an “effective amount” of an agent that blocks aTGF-β pathway is an amount that is effective to increase cognitivefunction, e.g., in an individual having reduced cognitive function as aresult of a neurodegenerative disorder such as Alzheimer's Disease (AD).In some embodiments, an “effective amount” of an agent that blocks aTGF-β pathway is an amount that is effective to increase cognitivefunction in an individual having reduced cognitive function as a resultof AD by at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 2-fold, at least about 5-fold, or at least about 10-fold, ormore, compared to the cognitive function in the individual in theabsence of treatment with the agent.

A subject method is suitable for treating various disorders including,e.g., epilepsy, traumatic brain injury, stroke, and neurodegenerativediseases. A subject method is suitable for treating epilepsy (includingposttraumatic epilepsy and post-ischemic epilepsy (i.e. after stroke),Parkinson's disease, psychosis, migraine, cerebral ischemic, Alzheimer'sdisease and other degenerative diseases such as neurological deficitsassociated with acquired immunodeficiency syndrome, traumatic braininjury, inappropriate neuronal activity resulting in neurodysthesias indiseases such as diabetes, neurological complications of diabetesmellitus, multiple sclerosis (MS) and motor neuron disease, ataxias,muscular rigidity (spasticity), and amyotrophic lateral sclerosis (ALS).

TGF-β Pathway Blockers

Suitable agents that block a TGF-β pathway include, e.g., TGF-β receptor(TGF-β-R) antagonists; agents that inhibit the activity of a TGF-βpathway element downstream of a TGF-β-R; and agents that reduce thelevel of a TGF-β pathway element downstream of a TGF-β-R. TGF-β pathwayinhibitors include, e.g., agents that inhibit phosphorylation of aTGF-β-R; agents that inhibit a kinase activity of a TGF-β-R; agents thatinhibit phosphorylation of a TGF-β pathway element downstream of aTGF-β-R; and the like. TGF-β pathway elements that are downstream of aTGF-β-R and that are targets of therapeutic agents as described hereininclude, e.g., NFκB, Smad1, Smad2, Smad6, Stat3, Stat1, Jak1, MAPK,Noggin, Thbs1 (thrombospondin 1), bone morphogenic protein-4 (BMP4),bone morphogenic protein-6 (BMP6), spp-1 (secreted phosphoprotein 1),Pal-1, TGFB-induced factor homeobox 1 (Tgif1), Tumor necrosis factor(TNF), and ENG.

TGF-β Receptor Antagonists

Suitable TGF-β-R antagonists include inhibitors of kinase activity of aTGF-β-R. TGF-β receptors include, e.g., TGF-βI, TGF-β-II, ALK1, andALK5.

Suitable TGF-β-R antagonists include, e.g., a Sm2 peptide as disclosedin U.S. Patent Publication No. 2005/0136043; inhibitors of aldosterone;anti-TGFβ antibodies, renin inhibitors, angiotensin converting enzyme(ACE) inhibitors; angiotensin II (AII) receptor antagonists;anti-TGF-β-R antibodies; and proteoglycans. Proteoglycans include, e.g.,decorin, biglycan, fibromodulin, lumican, betaglycan and endoglin.

Aldosterone inhibitors include, e.g., eplerenone (Inspra™):(7α,11α,17α)-pregn-4-ene-7,21-dicarboxylicacid,9,11-epoxy-17-hydroxy-3-oxo-,γ-lactone, methyl ester and compoundsrelated thereto (U.S. Pat. No. 4,559,332); spironolactone (Aldactone™):7α-acetylthio-3-oxo-17α-pregn-4-ene-21,17-carbolactone and compoundsrelated thereto; and a compound as described in, e.g., U.S. Pat. No.6,410,524.

AII receptor antagonists include, e.g., losartan (Cozaar™):2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-methanol,monopotassium salt and the various substituted imidazole derivatives andother compounds related thereto (see, e.g., U.S. Pat. No. 5,138,069; andWO 2007/020533); valsartan, (Diovan™):N-[p-(o-1H-tetrazol-5-yl-phenyl)benzyl]-N-valeryl-L-valine and compoundsrelated thereto (U.S. Pat. No. 5,399,578); irbesartan (Avapro™):2-n-butyl-4-spirocyclopentane-1-((2′-tetrazol-5-yl)biphenyl-4-yl)-2-imidazolin-5-oneand compounds related thereto (U.S. Pat. Nos. 5,270,317 and 5,352,788);candesartan (Amias™, Atacand™):1-(cyclohexyloxycarbonyloxy)ethyl-2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazole-7-carboxylateand compounds related thereto (in U.S. Pat. No. 5,196,444); telmisartan(Micardis™):4′-[(1,4′-dimethyl-2′-propyl[2,6-bi-1H-benzimidazol]-1-yl)methyl]-[1,1′-biphenyl]-2-carboxylicacid and compounds related thereto (European Pat. Application No.6502314); tasosartan (Verdia™):5,8-dihydro-2,4-dimethyl-8-[p-(o-1H-tetrazol-5-ylphenyl]pyrido[2,3-d]pyrimidin-7(6H)-oneand compounds related thereto (U.S. Pat. No. 5,149,699); eprosartan(Teveten™):4-({2-butyl-5-[2-carboxy-2-(thiophen-2-ylmethyl)eth-1-en-1-yl]-1H-imidazol-1-yl}methyl)benzoicacid and compounds related thereto (U.S. Pat. No. 5,185,351); saralasin:1-(N-methylglycine)-5-L-valine-8-L-alanineangiotensin II (an octapeptideanalog of Ang II (bovine) with amino acids 1 and 8 replaced withsarcosine and alanine, respectively; and a compound as disclosed in,e.g., U.S. Pat. Nos. 5,484,780; 6,028,091; and 6,329,384.

ACE inhibitors include, e.g., benazepril (Lotensin™, Lotrel™):3-[[1-(ethoxy-carbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-aceticacid monohydrochloride and its metabolite benazeprilat and compoundsrelated thereto (U.S. Pat. No. 4,410,520); captopril (Capoten™):1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline and compounds relatedthereto (U.S. Pat. No. 4,105,776); enalapril (Vasotec™):1-[N-[(S)-1-carboxy-3-phenylpropyl]-L-alanyl]-L-proline-1′-ethyl ester;lisinopril (Zestril™,1-[N.sub.2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl]-L-proline and thevarious carboxyalkyl dipeptide derivatives and compounds related thereto(U.S. Pat. Nos. 4,374,829, 6,468,976, and 6,465,615); perindoprilerbumine (Aceon™, Coversyl™):(2S,3αS,7αS)-1-[(S)-N-[(S)-1-carboxy-butyl]alanyl]hexahydro-2-indolinecarboxylicacid, 1-ethyl ester and compounds related thereto; quinapril(Accupril™):(3S)-2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-1,2,3,4-tetrahydro-isoquinoline-3-carboxyliquemonochlorhydrate and compounds related thereto; ramipril (Altace™):(2S,3αS,6αS)-1-[(S)-N-[(S)-1-carboxy-3-phenylpropyl]alanyl]octahydrocyclopenta[β]pyrrole-2-carboxylicacid, 1-ethyl ester and compounds related thereto; trandolapril(Mavik™):(2S,3αR,7αS)-1-[(S)-N-[(S)-Carboxy-3-phenylpropyl]alanyl]hexa-hydro-2-indolinecarboxylicacid, 1-ethyl ester and compounds related thereto; fosinopril(Monopril™): L-proline,4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy)propoxyl](4-phenylbutyl)phosphinyl]acetyl]sodium salt, trans-, and compounds related thereto; moexipril(Univasc™):3S-[2[R*(R*)],3R*]]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquinolinecarboxylicacid monohydrochloride and compounds related thereto; and imidapril(Tanatril™):(−)-4S)-3-[(2S)-2-[[(1S)-1-ethoxycarbonyl-3-phenylpropyl]amino-1-propionyl-]-1-methyl-2-oxoimidazolidine-4-carboxylicacid hydrochloride and compounds related thereto.

Renin inhibitors include, e.g., aliskiren (SPP100):2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-octanamidhemifumarate and compounds related thereto as disclosed in U.S. Pat. No.5,719,141 and WO 01/09079; enalkiren:[1S-(1R*,2S*,3R*)]-N-(3-amino-3-methyl-1-oxobutyl)-O-methyl-L-tyrosyl-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5-methylhexyl]-L-histidinamideand compound related thereto; and remikiren:(S)-2-tert-Butylsulphonylmethyl-N-[(S)-1-[(1S,2R,3S)-1-cyclo-hexylmethyl-3-cyclopropyl-2,3-dihydroxypropylcarbamoyl]-2-(1H-imidazol-4-yl)methyl]-3-phenylylpropionamideand compound related thereto.

Suitable TGF-β pathway inhibitors include selective inhibitors ofTGF-β-RII.

NFκB Inhibitors

Suitable NKκB inhibitors include, e.g., caffeic acid phenylethyl ester(CAPE), DM-CAPE, SN-50 peptide, hymenialdisine, and pyrrolidonedithiocarbamate.

MAPK Inhibitors

Mitogen-activated protein kinase (MAPK) inhibitor compounds suitable forthe invention include, for example,4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole(SB203580),4-(3-Iodophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole(SB203580-iodo),4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole(SB202190),5-(2-amino-4-pyrimidyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole(SB220025),4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316), and 2′-amino-3′-methoxyflavone (PD98059).

Smad Inhibitors

Suitable Smad inhibitors include, e.g., A-83-01(3-(6-Methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole;Alk-5 inhibitor, Masayoshi et al, 2005), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine; Alk-5 inhibitor,Sawyer et al, 2003), and SB-431542(4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide).Also suitable for use is a pyrimidine derivative as described in WO2008/006583.

Interfering Nucleic Acids

In some embodiments, an agent that inhibits TGF-β signaling is aninhibitory (or “interfering”) nucleic acid. Interfering nucleic acids(RNAi) include nucleic acids that provide for decreased levels of aTGF-β pathway element in a cell, e.g., a neuronal cell. Interferingnucleic acids include, e.g., a short interfering nucleic acid (siNA), ashort interfering RNA (siRNA), a double-stranded RNA (dsRNA), amicro-RNA (miRNA), and a short hairpin RNA (shRNA) molecule.

The term “short interfering nucleic acid,” “siNA,” “short interferingRNA,” “siRNA,” “short interfering nucleic acid molecule,” “shortinterfering oligonucleotide molecule,” or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expression,for example by mediating RNA interference “RNAi” or gene silencing in asequence-specific manner. Design of RNAi molecules when given a targetgene is routine in the art. See also US 2005/0282188 (which isincorporated herein by reference) as well as references cited therein.See, e.g., Pushparaj et al. Clin Exp Pharmacol Physiol. 2006 May-June;33(5-6):504-10; Lutzelberger et al. Handb Exp Pharmacol. 2006;(173):243-59; Aronin et al. Gene Ther. 2006 March; 13(6):509-16; Xie etal. Drug Discov Today. 2006 January; 11(1-2):67-73; Grunweller et al.Curr Med Chem. 2005; 12(26):3143-61; and Pekaraik et al. Brain Res Bull.2005 Dec. 15; 68(1-2):115-20. Epub 2005 September 9.

Methods for design and production of siRNAs to a desired target areknown in the art, and their application to TGF-β pathwayelement-encoding nucleic acids will be readily apparent to theordinarily skilled artisan, as are methods of production of siRNAshaving modifications (e.g., chemical modifications) to provide for,e.g., enhanced stability, bioavailability, and other properties toenhance use as therapeutics. In addition, methods for formulation anddelivery of siRNAs to a subject are also well known in the art. See,e.g., US 2005/0282188; US 2005/0239731; US 2005/0234232; US2005/0176018; US 2005/0059817; US 2005/0020525; US 2004/0192626; US2003/0073640; US 2002/0150936; US 2002/0142980; and US2002/0120129, eachof which are incorporated herein by reference.

Publicly available tools to facilitate design of siRNAs are available inthe art. See, e.g., DEQOR: Design and Quality Control of RNAi (availableon the internet at cluster-Lmpi-cbg.de/Deqor/deqor.html). See also,Henschel et al. Nucleic Acids Res. 2004 Jul. 1; 32(Web Serverissue):W113-20. DEQOR is a web-based program which uses a scoring systembased on state-of-the-art parameters for siRNA design to evaluate theinhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i)regions in a gene that show high silencing capacity based on the basepair composition and (ii) siRNAs with high silencing potential forchemical synthesis. In addition, each siRNA arising from the input queryis evaluated for possible cross-silencing activities by performing BLASTsearches against the transcriptome or genome of a selected organism.DEQOR can therefore predict the probability that an mRNA fragment willcross-react with other genes in the cell and helps researchers to designexperiments to test the specificity of siRNAs or chemically designedsiRNAs.

siNA molecules can be of any of a variety of forms. For example the siNAcan be a double-stranded polynucleotide molecule comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof. siNA can also be assembledfrom two separate oligonucleotides, where one strand is the sense strandand the other is the antisense strand, wherein the antisense and sensestrands are self-complementary. In this embodiment, each strandgenerally comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 15 base pairs toabout 30 base pairs, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15 nucleotides to about 25 ormore nucleotides of the siNA molecule are complementary to the targetnucleic acid or a portion thereof).

Alternatively, the siNA can be assembled from a single oligonucleotide,where the self-complementary sense and antisense regions of the siNA arelinked by a nucleic acid-based or non-nucleic acid-based linker(s). ThesiNA can be a polynucleotide with a duplex, asymmetric duplex, hairpinor asymmetric hairpin secondary structure, having self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in aseparate target nucleic acid molecule or a portion thereof and the senseregion having nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof.

The siNA can be a circular single-stranded polynucleotide having two ormore loop structures and a stem comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof, and wherein the circular polynucleotide can beprocessed either in vivo or in vitro to generate an active siNA moleculecapable of mediating RNAi. The siNA can also comprise a single strandedpolynucleotide having nucleotide sequence complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof (e.g.,where such siNA molecule does not require the presence within the siNAmolecule of nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof), wherein the single strandedpolynucleotide can further comprise a terminal phosphate group, such asa 5′-phosphate (see for example Martinez et al., 2002, Cell., 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or5′,3′-diphosphate.

In certain embodiments, the siNA molecule contains separate sense andantisense sequences or regions, wherein the sense and antisense regionsare covalently linked by nucleotide or non-nucleotide linkers moleculesas is known in the art, or are alternately non-covalently linked byionic interactions, hydrogen bonding, van der Waals interactions,hydrophobic interactions, and/or stacking interactions. In certainembodiments, the siNA molecules comprise nucleotide sequence that iscomplementary to nucleotide sequence of a target gene. In anotherembodiment, the siNA molecule interacts with nucleotide sequence of atarget gene in a manner that causes inhibition of expression of thetarget gene.

As used herein, siNA molecules need not be limited to those moleculescontaining only RNA, but further encompasses chemically-modifiednucleotides and non-nucleotides. In certain embodiments, the shortinterfering nucleic acid molecules of the invention lack 2′-hydroxy(2′-OH) containing nucleotides. siNAs do not necessarily require thepresence of nucleotides having a 2′-hydroxy group for mediating RNAi andas such, siNA molecules of the invention optionally do not include anyribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNAmolecules that do not require the presence of ribonucleotides within thesiNA molecule to support RNAi can however have an attached linker orlinkers or other attached or associated groups, moieties, or chainscontaining one or more nucleotides with 2′-OH groups. Optionally, siNAmolecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or50% of the nucleotide positions. The modified short interfering nucleicacid molecules of the invention can also be referred to as shortinterfering modified oligonucleotides “siMON.”

As used herein, the term siNA is meant to be equivalent to other termsused to describe nucleic acid molecules that are capable of mediatingsequence specific RNAi, for example short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA(shRNA), short interfering oligonucleotide, short interfering nucleicacid, short interfering modified oligonucleotide, chemically-modifiedsiRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term RNAi is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics. For example, siNA molecules of the invention can be used toepigenetically silence a target gene at the post-transcriptional leveland/or at the pre-transcriptional level. In a non-limiting example,epigenetic regulation of gene expression by siNA molecules of theinvention can result from siNA mediated modification of chromatinstructure or methylation pattern to alter gene expression (see, forexample, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al.,2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

siNA molecules contemplated herein can comprise a duplex formingoligonucleotide (DFO) see, e.g., WO 05/019453; and US 2005/0233329,which are incorporated herein by reference). siNA molecules alsocontemplated herein include multifunctional siNA, (see, e.g., WO05/019453 and US 2004/0249178). The multifunctional siNA can comprisesequence targeting, for example, two regions of Skp2.

siNA molecules contemplated herein can comprise an asymmetric hairpin orasymmetric duplex. By “asymmetric hairpin” as used herein is meant alinear siNA molecule comprising an antisense region, a loop portion thatcan comprise nucleotides or non-nucleotides, and a sense region thatcomprises fewer nucleotides than the antisense region to the extent thatthe sense region has enough complementary nucleotides to base pair withthe antisense region and form a duplex with loop. For example, anasymmetric hairpin siNA molecule can comprise an antisense region havinglength sufficient to mediate RNAi in a cell or in vitro system (e.g.about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

Stability and/or half-life of siRNAs can be improved through chemicallysynthesizing nucleic acid molecules with modifications (base, sugarand/or phosphate) can prevent their degradation by serum ribonucleases,which can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat.No. 6,300,074; and Burgin et al., supra; all of which are incorporatedby reference herein, describing various chemical modifications that canbe made to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

For example, oligonucleotides are modified to enhance stability and/orenhance biological activity by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl,2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Eamshaw and Gait, 1998, Biopolymers (Nucleic AcidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; eachof which are hereby incorporated in their totality by reference herein).In view of such teachings, similar modifications can be used asdescribed herein to modify the siNA nucleic acid molecules of disclosedherein so long as the ability of siNA to promote RNAi is cells is notsignificantly inhibited.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are contemplated herein.Such a nucleic acid is also generally more resistant to nucleases thanan unmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. Nucleic acid moleculesdelivered exogenously are generally selected to be stable within cellsat least for a period sufficient for transcription and/or translation ofthe target RNA to occur and to provide for modulation of production ofthe encoded mRNA and/or polypeptide so as to facilitate reduction of thelevel of the target gene product.

Production of RNA and DNA molecules can be accomplished syntheticallyand can provide for introduction of nucleotide modifications to providefor enhanced nuclease stability. (see, e.g., Wincott et al., 1995,Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods inEnzymology 211, 3-19, incorporated by reference herein. In oneembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides, which are modified cytosine analogs which confer theability to hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine within a duplex, and can provide for enhancedaffinity and specificity to nucleic acid targets (see, e.g., Lin et al.1998, J. Am. Chem. Soc., 120, 8531-8532). In another example, nucleicacid molecules can include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a2′,4′-C methylene bicyclo nucleotide (see, e.g., Wengel et al., WO00/66604 and WO 99/14226).

siNA molecules can be provided as conjugates and/or complexes, e.g., tofacilitate delivery of siNA molecules into a cell. Exemplary conjugatesand/or complexes include those composed of an siNA and a small molecule,lipid, cholesterol, phospholipid, nucleoside, antibody, toxin,negatively charged polymer (e.g., protein, peptide, hormone,carbohydrate, polyethylene glycol, or polyamine). In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds can improve delivery and/or localization of nucleic acidmolecules into cells in the presence or absence of serum (see, e.g.,U.S. Pat. No. 5,854,038). Conjugates of the molecules described hereincan be attached to biologically active molecules via linkers that arebiodegradable, such as biodegradable nucleic acid linker molecules.

BBB Disruption

In some embodiments, an individual who is being considered for treatmentwith a subject method is first assessed for blood-brain barrier (BBB)disruption. Where the individual is determined to have BBB disruption,the individual is treated with an effective amount of an agent thatinhibits a TGF-β pathway. Methods of determining whether an individualhas BBB disruption are known in the art.

Formulations, Dosages, and Routes of Administration

An agent that blocks a TGF-β pathway can be provided together with apharmaceutically acceptable excipient. Pharmaceutically acceptableexcipients are known to those skilled in the art, and have been amplydescribed in a variety of publications, including, for example, A.Gennaro (1995) “Remington: The Science and Practice of Pharmacy”, 19thedition, Lippincott, Williams, & Wilkins

Formulations

An agent that blocks a TGF-β pathway is also referred to herein as an“active agent,” “agent,” or “drug.” In the subject methods, the activeagent(s) may be administered to the host using any convenient meanscapable of resulting in the desired reduction in disease symptoms.

An active agent can be incorporated into a variety of formulations fortherapeutic administration. More particularly, an active agent can beformulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, an active agent may be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Anactive agent can be administered rectally via a suppository. Thesuppository can include vehicles such as cocoa butter, carbowaxes andpolyethylene glycols, which melt at body temperature, yet are solidifiedat room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more activeagents. Similarly, unit dosage forms for injection or intravenousadministration may comprise the agent(s) in a composition as a solutionin sterile water, normal saline or another pharmaceutically acceptablecarrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of an activeagent calculated in an amount sufficient to produce the desired effectin association with a pharmaceutically acceptable diluent, carrier orvehicle. The specifications for the active agents depend on theparticular compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

Other modes of administration will also find use with the subjectinvention. For instance, an active agent can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), or about 1% toabout 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

An active agent can be administered as injectables. Typically,injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985; Remington: The Science and Practice of Pharmacy, A.R.Gennaro, (2000) Lippincott, Williams & Wilkins. The composition orformulation to be administered will, in any event, contain a quantity ofthe agent adequate to achieve the desired state in the subject beingtreated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Oral Formulations

In some embodiments, an active agent is formulated for oral delivery toan individual in need of such an agent.

For oral delivery, a subject formulation comprising an active agent willin some embodiments include an enteric-soluble coating material.Suitable enteric-soluble coating material include hydroxypropylmethylcellulose acetate succinate (HPMCAS), hydroxypropyl methylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),polyvinyl phthalic acetate (PVPA), Eudragit™, and shellac.

As one non-limiting example of a suitable oral formulation, an activeagent is formulated with one or more pharmaceutical excipients andcoated with an enteric coating, as described in U.S. Pat. No. 6,346,269.For example, a solution comprising an active agent and a stabilizer iscoated onto a core comprising pharmaceutically acceptable excipients, toform an active agent-coated core; a sub-coating layer is applied to theactive agent-coated core, which is then coated with an enteric coatinglayer. The core generally includes pharmaceutically inactive componentssuch as lactose, a starch, mannitol, sodium carboxymethyl cellulose,sodium starch glycolate, sodium chloride, potassium chloride, pigments,salts of alginic acid, talc, titanium dioxide, stearic acid, stearate,micro-crystalline cellulose, glycerin, polyethylene glycol, triethylcitrate, tributyl citrate, propanyl triacetate, dibasic calciumphosphate, tribasic sodium phosphate, calcium sulfate, cyclodextrin, andcastor oil. Suitable solvents for the active agent include aqueoussolvents. Suitable stabilizers include alkali-metals and alkaline earthmetals, bases of phosphates and organic acid salts and organic amines.The sub-coating layer comprises one or more of an adhesive, aplasticizer, and an anti-tackiness agent. Suitable anti-tackiness agentsinclude talc, stearic acid, stearate, sodium stearyl fumarate, glycerylbehenate, kaolin and aerosil. Suitable adhesives include polyvinylpyrrolidone (PVP), gelatin, hydroxyethyl cellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC), vinyl acetate(VA), polyvinyl alcohol (PVA), methyl cellulose (MC), ethyl cellulose(EC), hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalates (CAP), xanthan gum, alginic acid, salts of alginicacid, Eudragit™, copolymer of methyl acrylic acid/methyl methacrylatewith polyvinyl acetate phthalate (PVAP). Suitable plasticizers includeglycerin, polyethylene glycol, triethyl citrate, tributyl citrate,propanyl triacetate and castor oil. Suitable enteric-soluble coatingmaterial include hydroxypropyl methylcellulose acetate succinate(HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalate (CAP), polyvinyl phthalic acetate (PVPA), Eudragit™and shellac.

Suitable oral formulations also include an active agent, formulated withany of the following: microgranules (see, e.g., U.S. Pat. No.6,458,398); biodegradable macromers (see, e.g., U.S. Pat. No.6,703,037); biodegradable hydrogels (see, e.g., Graham and McNeill(1989) Biomaterials 5:27-36); biodegradable particulate vectors (see,e.g., U.S. Pat. No. 5,736,371); bioabsorbable lactone polymers (see,e.g., U.S. Pat. No. 5,631,015); slow release protein polymers (see,e.g., U.S. Pat. No. 6,699,504; Pelias Technologies, Inc.); apoly(lactide-co-glycolide/polyethylene glycol block copolymer (see,e.g., U.S. Pat. No. 6,630,155; Atrix Laboratories, Inc.); a compositioncomprising a biocompatible polymer and particles of metalcation-stabilized agent dispersed within the polymer (see, e.g., U.S.Pat. No. 6,379,701; Alkermes Controlled Therapeutics, Inc.); andmicrospheres (see, e.g., U.S. Pat. No. 6,303,148; Octoplus, B.V.).

Suitable oral formulations also include an active agent formulated withany of the following: a carrier such as Emisphere® (EmisphereTechnologies, Inc.); TIMER^(x), a hydrophilic matrix combining xanthanand locust bean gums which, in the presence of dextrose, form a strongbinder gel in water (Penwest); Geminex™ (Penwest); Procise™(GlaxoSmithKline); SAVIT™ (Mistral Pharma Inc.); RingCap™ (Alza Corp.);Smartrix® (Smartrix Technologies, Inc.); SQZge1™ (MacroMed, Inc.);Geomatrix™ (Skye Pharma, Inc.); Oros® Tri-layer (Alza Corporation); andthe like.

Also suitable for use are formulations such as those described in U.S.Pat. No. 6,296,842 (Alkermes Controlled Therapeutics, Inc.); U.S. Pat.No. 6,187,330 (Scios, Inc.); and the like.

Also suitable for use herein are formulations comprising an intestinalabsorption enhancing agent. Suitable intestinal absorption enhancersinclude, but are not limited to, calcium chelators (e.g., citrate,ethylenediamine tetracetic acid); surfactants (e.g., sodium dodecylsulfate, bile salts, palmitoylcarnitine, and sodium salts of fattyacids); toxins (e.g., zonula occludens toxin); and the like.

Controlled Release Formulations

In some embodiments, an active agent is formulated in a controlledrelease formulation.

Controlled release within the scope of this invention can be taken tomean any one of a number of extended release dosage forms. The followingterms may be considered to be substantially equivalent to controlledrelease, for the purposes of the present invention: continuous release,controlled release, delayed release, depot, gradual release, long-termrelease, programmed release, prolonged release, proportionate release,protracted release, repository, retard, slow release, spaced release,sustained release, time coat, timed release, delayed action, extendedaction, layered-time action, long acting, prolonged action, repeatedaction, slowing acting, sustained action, sustained-action medications,and extended release. Further discussions of these terms may be found inLesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press,Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems.

Physical systems include, but are not limited to, reservoir systems withrate-controlling membranes, such as microencapsulation,macroencapsulation, and membrane systems; reservoir systems withoutrate-controlling membranes, such as hollow fibers, ultra microporouscellulose triacetate, and porous polymeric substrates and foams;monolithic systems, including those systems physically dissolved innon-porous, polymeric, or elastomeric matrices (e.g., nonerodible,erodible, environmental agent ingression, and degradable), and materialsphysically dispersed in non-porous, polymeric, or elastomeric matrices(e.g., nonerodible, erodible, environmental agent ingression, anddegradable); laminated structures, including reservoir layers chemicallysimilar or dissimilar to outer control layers; and other physicalmethods, such as osmotic pumps, or adsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release may be found in Agis F. Kydonieus, Controlled ReleaseTechnologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

There are a number of controlled release drug formulations that aredeveloped for oral administration. These include, but are not limitedto, osmotic pressure-controlled gastrointestinal delivery systems;hydrodynamic pressure-controlled gastrointestinal delivery systems;membrane permeation-controlled gastrointestinal delivery systems, whichinclude microporous membrane permeation-controlled gastrointestinaldelivery devices; gastric fluid-resistant intestine targetedcontrolled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems may be found in Yie W. Chien, Novel DrugDelivery Systems, 1992 (Marcel Dekker, Inc.). Some of these formulationswill now be discussed in more detail.

Enteric coatings are applied to tablets to prevent the release of drugsin the stomach either to reduce the risk of unpleasant side effects orto maintain the stability of the drug which might otherwise be subjectto degradation of expose to the gastric environment. Most polymers thatare used for this purpose are polyacids that function by virtue or thefact that their solubility in aqueous medium is pH-dependent, and theyrequire conditions with a pH higher than normally encountered in thestomach.

One exemplary type of oral controlled release structure is entericcoating of a solid or liquid dosage form. The enteric coatings aredesigned to disintegrate in intestinal fluid for ready absorption. Delayof absorption of the active agent that is incorporated into aformulation with an enteric coating is dependent on the rate of transferthrough the gastrointestinal tract, and so the rate of gastric emptyingis an important factor. Some investigators have reported that amultiple-unit type dosage form, such as granules, may be superior to asingle-unit type. Therefore, in one exemplary embodiment, an activeagent is contained in an enterically coated multiple-unit dosage form.In an exemplary embodiment, an active agent dosage form is prepared byspray-coating granules of an active agent-enteric coating agent soliddispersion on an inert core material. These granules can result inprolonged absorption of the drug with good bioavailability.

Suitable enteric coating agents include, but are not limited to,hydroxypropylmethylcellulose phthalate, methacryclic acid-methacrylicacid ester copolymer, polyvinyl acetate-phthalate and cellulose acetatephthalate Akihiko Hasegawa, Application of solid dispersions ofNifedipine with enteric coating agent to prepare a sustained-releasedosage form, Chem. Pharm. Bull. 33: 1615-1619 (1985). Various entericcoating materials may be selected on the basis of testing to achieve anenteric coated dosage form designed ab initio to have an optimalcombination of dissolution time, coating thicknesses and diametralcrushing strength. S. C. Porter et al., The Properties of Enteric TabletCoatings Made From Polyvinyl Acetate-phthalate and Cellulose acetatePhthalate, J. Pharm. Pharmacol. 22:42p (1970).

Another type of useful oral controlled release structure is a soliddispersion. A solid dispersion may be defined as a dispersion of one ormore active ingredients in an inert carrier or matrix in the solid stateprepared by the melting (fusion), solvent, or melting-solvent methodAkihiko Hasegawa, Super Saturation Mechanism of Drugs from SolidDispersions with Enteric Coating Agents, Chem. Pharm. Bull. 36:4941-4950 (1998). The solid dispersions may be also called solid-statedispersions. The term “coprecipitates” may also be used to refer tothose preparations obtained by the solvent methods.

The selection of the carrier may have an influence on the dissolutioncharacteristics of the dispersed drug (e.g., active agent) because thedissolution rate of a component from a surface may be affected by othercomponents in a multiple component mixture. For example, a water-solublecarrier may result in a fast release of the drug from the matrix, or apoorly soluble or insoluble carrier may lead to a slower release of thedrug from the matrix. The solubility of the active agent may also beincreased owing to some interaction with the carriers.

Examples of carriers useful in solid dispersions include, but are notlimited to, water-soluble polymers such as polyethylene glycol,polyvinylpyraolidone, and hydroxypropylmethyl-cellulose. Alternativecarriers include phosphatidylcholine. Phosphatidylcholine is anamphoteric but water-insoluble lipid, which may improve the solubilityof otherwise insoluble active agents in an amorphous state inphosphatidylcholine solid dispersions.

Other carriers include polyoxyethylene hydrogenated castor oil. Poorlywater-soluble active agents may be included in a solid dispersion systemwith an enteric polymer such as hydroxypropylmethylcellulose phthalateand carboxymethylethylcellulose, and a non-enteric polymer,hydroxypropylmethylcellulose. Another solid dispersion dosage formincludes incorporation of the drug of interest (e.g., an active agent)with ethyl cellulose and stearic acid in different ratios.

There are various methods commonly known for preparing soliddispersions. These include, but are not limited to, the melting method,the solvent method and the melting-solvent method.

Another controlled release dosage form is a complex between an ionexchange resin and an active agent. Ion exchange resin-drug complexeshave been used to formulate sustained-release products of acidic andbasic drugs. In one exemplary embodiment, a polymeric film coating isprovided to the ion exchange resin-drug complex particles, making drugrelease from these particles diffusion controlled. See Y. Raghunathan etal., Sustained-released drug delivery system I: Coded ion-exchange resinsystems for phenylpropanolamine and other drugs, J. Pharm. Sciences 70:379-384 (1981).

Injectable microspheres are another controlled release dosage form.Injectable micro spheres may be prepared by non-aqueous phase separationtechniques, and spray-drying techniques. Microspheres may be preparedusing polylactic acid or copoly(lactic/glycolic acid). Shigeyuki Takada,Utilization of an Amorphous Form of a Water-Soluble GPIIb/IIIaAntagonist for Controlled Release From Biodegradable Micro spheres,Pharm. Res. 14:1146-1150 (1997), and ethyl cellulose, Yoshiyuki Koida,Studies on Dissolution Mechanism of Drugs from Ethyl CelluloseMicrocapsules, Chem. Pharm. Bull. 35:1538-1545 (1987).

Other controlled release technologies that may be used include, but arenot limited to, SODAS (Spheroidal Oral Drug Absorption System), INDAS(Insoluble Drug Absorption System), IPDAS (Intestinal Protective DrugAbsorption System), MODAS (Multiporous Oral Drug Absorption System),EFVAS (Effervescent Drug Absorption System), PRODAS (Programmable OralDrug Absorption System), and DUREDAS (Dual Release Drug AbsorptionSystem) available from Elan Pharmaceutical Technologies. SODAS are multiparticulate dosage forms utilizing controlled release beads. INDAS are afamily of drug delivery technologies designed to increase the solubilityof poorly soluble drugs. IPDAS are multi particulate tablet formationutilizing a combination of high density controlled release beads and animmediate release granulate. MODAS are controlled release single unitdosage forms. Each tablet consists of an inner core surrounded by asemipermeable multiparous membrane that controls the rate of drugrelease. EFVAS is an effervescent drug absorption system. PRODAS is afamily of multi particulate formulations utilizing combinations ofimmediate release and controlled release mini-tablets. DUREDAS is abilayer tablet formulation providing dual release rates within the onedosage form. Although these dosage forms are known to one of skill,certain of these dosage forms will now be discussed in more detail.

INDAS was developed specifically to improve the solubility andabsorption characteristics of poorly water soluble drugs. Solubilityand, in particular, dissolution within the fluids of thegastrointestinal tract is a key factor in determining the overall oralbioavailability of poorly water soluble drug. By enhancing solubility,one can increase the overall bioavailability of a drug with resultingreductions in dosage. INDAS takes the form of a high energy matrixtablet, production of which is comprised of two distinct steps: the drugin question is converted to an amorphous form through a combination ofenergy, excipients, and unique processing procedures.

Once converted to the desirable physical form, the resultant high energycomplex may be stabilized by an absorption process that utilizes a novelpolymer cross-linked technology to prevent recrystallization. Thecombination of the change in the physical state of the active agentcoupled with the solubilizing characteristics of the excipients employedenhances the solubility of the active agent. The resulting absorbedamorphous drug complex granulate may be formulated with a gel-formingerodible tablet system to promote substantially smooth and continuousabsorption.

IPDAS is a multi-particulate tablet technology that may enhance thegastrointestinal tolerability of potential irritant and ulcerogenicdrugs. Intestinal protection is facilitated by the multi-particulatenature of the IPDAS formulation which promotes dispersion of an irritantlipoate throughout the gastrointestinal tract. Controlled releasecharacteristics of the individual beads may avoid high concentration ofdrug being both released locally and absorbed systemically. Thecombination of both approaches serves to minimize the potential harm ofan active agent with resultant benefits to patients.

IPDAS is composed of numerous high density controlled release beads.Each bead may be manufactured by a two step process that involves theinitial production of a micromatrix with embedded active agent and thesubsequent coating of this micromatrix with polymer solutions that forma rate-limiting semipermeable membrane in vivo. Once an IPDAS tablet isingested, it may disintegrate and liberate the beads in the stomach.These beads may subsequently pass into the duodenum and along thegastrointestinal tract, e.g., in a controlled and gradual manner,independent of the feeding state. Release of the active agent occurs bydiffusion process through the micromatrix and subsequently through thepores in the rate controlling semipermeable membrane. The release ratefrom the IPDAS tablet may be customized to deliver a drug-specificabsorption profile associated with optimized clinical benefit. Should afast onset of activity be necessary, immediate-release granulate may beincluded in the tablet. The tablet may be broken prior toadministration, without substantially compromising drug release, if areduced dose is required for individual titration.

MODAS is a drug delivery system that may be used to control theabsorption of water soluble agents. Physically MODAS is anon-disintegrating table formulation that manipulates drug release by aprocess of rate limiting diffusion by a semipermeable membrane formed invivo. The diffusion process essentially dictates the rate ofpresentation of drug to the gastrointestinal fluids, such that theuptake into the body is controlled. Because of the minimal use ofexcipients, MODAS can readily accommodate small dosage size forms. EachMODAS tablet begins as a core containing active drug plus excipients.This core is coated with a solution of insoluble polymers and solubleexcipients. Once the tablet is ingested, the fluid of thegastrointestinal tract may dissolve the soluble excipients in the outercoating leaving substantially the insoluble polymer. What results is anetwork of tiny, narrow channels connecting fluid from thegastrointestinal tract to the inner drug core of water soluble drug.This fluid passes through these channels, into the core, dissolving thedrug, and the resultant solution of drug may diffuse out in a controlledmanner. This may permit both controlled dissolution and absorption. Anadvantage of this system is that the drug-releasing pores of the tabletare distributed over substantially the entire surface of the tablet.This facilitates uniform drug absorption reduces aggressiveunidirectional drug delivery. MODAS represents a very flexible dosageform in that both the inner core and the outer semipermeable membranemay be altered to suit the individual delivery requirements of a drug.In particular, the addition of excipients to the inner core may help toproduce a microenvironment within the tablet that facilitates morepredictable release and absorption rates. The addition of an immediaterelease outer coating may allow for development of combination products.

Additionally, PRODAS may be used to deliver an active agent. PRODAS is amulti particulate drug delivery technology based on the production ofcontrolled release mini tablets in the size range of 1.5 to 4 mm indiameter. The PRODAS technology is a hybrid of multi particulate andhydrophilic matrix tablet approaches, and may incorporate, in one dosageform, the benefits of both these drug delivery systems.

In its most basic form, PRODAS involves the direct compression of animmediate release granulate to produce individual mini tablets thatcontain an active agent. These mini tablets are subsequentlyincorporated into hard gels and capsules that represent the final dosageform. A more beneficial use of this technology is in the production ofcontrolled release formulations. In this case, the incorporation ofvarious polymer combinations within the granulate may delay the releaserate of drugs from each of the individual mini tablets. These minitablets may subsequently be coated with controlled release polymersolutions to provide additional delayed release properties. Theadditional coating may be necessary in the case of highly water solubledrugs or drugs that are perhaps gastroirritants where release can bedelayed until the formulation reaches more distal regions of thegastrointestinal tract. One value of PRODAS technology lies in theinherent flexibility to formulation whereby combinations of minitablets, each with different release rates, are incorporated into onedosage form. As well as potentially permitting controlled absorptionover a specific period, this also may permit targeted delivery of drugto specific sites of absorption throughout the gastrointestinal tract.Combination products also may be possible using mini tablets formulatedwith different active ingredients.

DUREDAS is a bilayer tableting technology that may be used to formulatean active agent. DUREDAS was developed to provide for two differentrelease rates, or dual release of a drug from one dosage form. The termbilayer refers to two separate direct compression events that take placeduring the tableting process. In an exemplary embodiment, an immediaterelease granulate is first compressed, being followed by the addition ofa controlled release element which is then compressed onto this initialtablet. This may give rise to the characteristic bilayer seen in thefinal dosage form.

The controlled release properties may be provided by a combination ofhydrophilic polymers. In certain cases, a rapid release of an activeagent may be desirable in order to facilitate a fast onset oftherapeutic affect. Hence one layer of the tablet may be formulated asan immediate-release granulate. By contrast, the second layer of thetablet may release the drug in a controlled manner, e.g., through theuse of hydrophilic polymers. This controlled release may result from acombination of diffusion and erosion through the hydrophilic polymermatrix.

A further extension of DUREDAS technology is the production ofcontrolled release combination dosage forms. In this instance, twodifferent active agents may be incorporated into the bilayer tablet andthe release of drug from each layer controlled to maximize therapeuticaffect of the combination.

An active agent can be incorporated into any one of the aforementionedcontrolled released dosage forms, or other conventional dosage forms.The amount of active agent contained in each dose can be adjusted, tomeet the needs of the individual patient, and the indication. One ofskill in the art and reading this disclosure will readily recognize howto adjust the level of an active agent and the release rates in acontrolled release formulation, in order to optimize delivery of anactive agent and its bioavailability.

Inhalational Formulations

An active agent will in some embodiments be administered to a patient bymeans of a pharmaceutical delivery system for the inhalation route. Theactive agent may be formulated in a form suitable for administration byinhalation. The inhalational route of administration provides theadvantage that the inhaled drug can bypass the blood-brain barrier. Thepharmaceutical delivery system is one that is suitable for respiratorytherapy by delivery of an active agent to mucosal linings of thebronchi. This invention can utilize a system that depends on the powerof a compressed gas to expel the active agent from a container. Anaerosol or pressurized package can be employed for this purpose.

As used herein, the term “aerosol” is used in its conventional sense asreferring to very fine liquid or solid particles carries by a propellantgas under pressure to a site of therapeutic application. When apharmaceutical aerosol is employed in this invention, the aerosolcontains the therapeutically active compound (e.g., active agent), whichcan be dissolved, suspended, or emulsified in a mixture of a fluidcarrier and a propellant. The aerosol can be in the form of a solution,suspension, emulsion, powder, or semi-solid preparation. Aerosolsemployed in the present invention are intended for administration asfine, solid particles or as liquid mists via the respiratory tract of apatient. Various types of propellants known to one of skill in the artcan be utilized. Suitable propellants include, but are not limited to,hydrocarbons or other suitable gas. In the case of the pressurizedaerosol, the dosage unit may be determined by providing a value todeliver a metered amount.

An active agent can also be formulated for delivery with a nebulizer,which is an instrument that generates very fine liquid particles ofsubstantially uniform size in a gas. For example, a liquid containingthe active agent is dispersed as droplets. The small droplets can becarried by a current of air through an outlet tube of the nebulizer. Theresulting mist penetrates into the respiratory tract of the patient.

A powder composition containing an active agent, with or without alubricant, carrier, or propellant, can be administered to a mammal inneed of therapy. This embodiment of the invention can be carried outwith a conventional device for administering a powder pharmaceuticalcomposition by inhalation. For example, a powder mixture of the compoundand a suitable powder base such as lactose or starch may be presented inunit dosage form in for example capsular or cartridges, e.g. gelatin, orblister packs, from which the powder may be administered with the aid ofan inhaler.

There are several different types of inhalation methodologies which canbe employed in connection with the present invention. An active agentcan be formulated in basically three different types of formulations forinhalation. First, an active agent can be formulated with low boilingpoint propellants. Such formulations are generally administered byconventional meter dose inhalers (MDI's). However, conventional MDI'scan be modified so as to increase the ability to obtain repeatabledosing by utilizing technology which measures the inspiratory volume andflow rate of the patient as discussed within U.S. Pat. Nos. 5,404,871and 5,542,410.

Alternatively, an active agent can be formulated in aqueous or ethanolicsolutions and delivered by conventional nebulizers. In some embodiments,such solution formulations are aerosolized using devices and systemssuch as disclosed within U.S. Pat. Nos. 5,497,763; 5,544,646; 5,718,222;and 5,660,166.

An active agent can be formulated into dry powder formulations. Suchformulations can be administered by simply inhaling the dry powderformulation after creating an aerosol mist of the powder. Technology forcarrying such out is described within U.S. Pat. No. 5,775,320 and U.S.Pat. No. 5,740,794.

Dosages

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of an agent that blocks a TGF-βpathway can be administered in a single dose. For example, a single doseof an active agent can include from about 1 ng to about 10 ng, fromabout 10 ng to about 25 ng, from about 25 ng to about 50 ng, from about50 ng to about 100 ng, from about 100 ng to about 500 ng, from about 500ng to about 1 mg, from about 1 mg to about 5 mg, or from about 5 mg toabout 10 mg, of active agent in a single dose. Alternatively, a targetdosage of agent that blocks a TGF-β pathway can be considered to beabout in the range of about 0.1-1000 μM, about 0.5-500 μM, about 1-100μM, or about 5-50 μM in a sample of host blood drawn within the first24-48 hours after administration of the agent.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

In some embodiments, multiple doses of an active agent are administered.The frequency of administration of an active agent can vary depending onany of a variety of factors, e.g., severity of the symptoms, etc. Forexample, in some embodiments, an active agent is administered once permonth, twice per month, three times per month, every other week (qow),once per week (qw), twice per week (biw), three times per week (tiw),four times per week, five times per week, six times per week, everyother day (qod), daily (qd), twice a day (qid), or three times a day(tid). In some embodiments, an active agent is administeredcontinuously.

The duration of administration of an active agent, e.g., the period oftime over which an active agent is administered, can vary, depending onany of a variety of factors, e.g., patient response, etc. For example,an active agent can be administered over a period of time ranging fromabout one day to about one week, from about two weeks to about fourweeks, from about one month to about two months, from about two monthsto about four months, from about four months to about six months, fromabout six months to about eight months, from about eight months to about1 year, from about 1 year to about 2 years, or from about 2 years toabout 4 years, or more. In some embodiments, an agent that blocks aTGF-β pathway is administered for the lifetime of the individual.

In some embodiments, administration of an active agent is discontinuous,e.g., an active agent is administered for a first period of time and ata first dosing frequency; administration of the active agent issuspended for a period of time; then the active agent is administeredfor a second period of time for a second dosing frequency. The period oftime during which administration of the active agent is suspended canvary depending on various factors, e.g., cognitive functions of theindividual; and will generally range from about 1 week to about 6months, e.g., from about 1 week to about 2 weeks, from about 2 weeks toabout 4 weeks, from about one month to about 2 months, from about 2months to about 4 months, or from about 4 months to about 6 months, orlonger. The first period of time may be the same or different than thesecond period of time; and the first dosing frequency may be the same ordifferent than the second dosing frequency.

Routes of Administration

An agent that blocks a TGF-β pathway is administered to an individualusing any available method and route suitable for drug delivery,including in vivo and ex vivo methods, as well as systemic and localizedroutes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, topical application, intravenous, rectal, nasal, oral andother parenteral routes of administration. Routes of administration maybe combined, if desired, or adjusted depending upon the agent and/or thedesired effect. The composition can be administered in a single dose orin multiple doses.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intracranial, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery ofthe agent. Where systemic delivery is desired, administration typicallyinvolves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

In some embodiments, an active agent is delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present invention. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of active agent can be accomplished usingany of a variety of refillable, pump systems. Pumps provide consistent,controlled release over time. In some embodiments, the agent is in aliquid formulation in a drug-impermeable reservoir, and is delivered ina continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use in the invention may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use with the present invention.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and thelike. In general, a subject treatment method can be accomplished usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems are generally preferred due to their generallymore consistent, controlled release over time. Osmotic pumps are used insome embodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCT publishedapplication no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and5,728,396)). Exemplary osmotically-driven devices suitable for use inthe invention include, but are not necessarily limited to, thosedescribed in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426;3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202;4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850;4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692;5,234,693; 5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted infra, animplantation site is a site within the body of a subject at which a drugdelivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, an active agent is delivered using an implantabledrug delivery system, e.g., a system that is programmable to provide foradministration of the agent. Exemplary programmable, implantable systemsinclude implantable infusion pumps. Exemplary implantable infusionpumps, or devices useful in connection with such pumps, are describedin, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019;5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and6,512,954. A further exemplary device that can be adapted for thepresent invention is the Synchromed infusion pump (Medtronic).

Crossing the Blood-Brain Barrier

The blood-brain barrier limits the uptake of many therapeutic agentsinto the brain and spinal cord from the general circulation. Moleculeswhich cross the blood-brain barrier use two main mechanisms: freediffusion; and facilitated transport. Because of the presence of theblood-brain barrier, attaining beneficial concentrations of a giventherapeutic agent in the central nervous system (CNS) may require theuse of drug delivery strategies. Delivery of therapeutic agents to theCNS can be achieved by several methods.

One method relies on neurosurgical techniques. In the case of gravelyill patients such as accident victims or those suffering from variousforms of dementia, surgical intervention is warranted despite itsattendant risks. For instance, therapeutic agents can be delivered bydirect physical introduction into the CNS, such as intraventricular orintrathecal injection of drugs. Intraventricular injection may befacilitated by an intraventricular catheter, for example, attached to areservoir, such as an Ommaya reservoir. Methods of introduction may alsobe provided by rechargeable or biodegradable devices. Another approachis the disruption of the blood-brain barrier by substances whichincrease the permeability of the blood-brain barrier. Examples includeintra-arterial infusion of poorly diffusible agents such as mannitol,pharmaceuticals which increase cerebrovascular permeability such asetoposide, or vasoactive agents such as leukotrienes. Neuwelt andRappoport (1984) Fed. Proc. 43:214-219; Baba et al. (1991) J. Cereb.Blood Flow Metab. 11:638-643; and Gennuso et al. (1993) Cancer Invest.11:638-643.

Further, it may be desirable to administer the pharmaceutical agentslocally to the area in need of treatment; this may be achieved by, forexample, local infusion during surgery, by injection, by means of acatheter, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers.

Therapeutic compounds can also be delivered by using pharmacologicaltechniques including chemical modification or screening for an analogwhich will cross the blood-brain barrier. The compound may be modifiedto increase the hydrophobicity of the molecule, decrease net charge ormolecular weight of the molecule, or modify the molecule, so that itwill resemble one normally transported across the blood-brain barrier.Levin (1980) J. Med. Chem. 23:682-684; Pardridge (1991) in: Peptide DrugDelivery to the Brain; and Kostis et al. (1994) J. Clin. Pharmacol.34:989-996.

Encapsulation of the drug in a hydrophobic environment such as liposomesis also effective in delivering drugs to the CNS. For example WO91/04014 describes a liposomal delivery system in which the drug isencapsulated within liposomes to which molecules have been added thatare normally transported across the blood-brain barrier.

Another method of formulating the drug to pass through the blood-brainbarrier is to encapsulate the drug in a cyclodextrin. Any suitablecyclodextrin which passes through the blood-brain barrier may beemployed, including, but not limited to, α-cyclodextrin, β-cyclodextrinand derivatives thereof. See generally, U.S. Pat. Nos. 5,017,566,5,002,935 and 4,983,586. Such compositions may also include a glycerolderivative as described by U.S. Pat. No. 5,153,179.

Delivery may also be obtained by conjugation of a therapeutic agent to atransportable agent to yield a new chimeric transportable therapeuticagent. For example, vasoactive intestinal peptide analog (VIPa) exertedits vasoactive effects only after conjugation to a monoclonal antibody(Mab) to the specific carrier molecule transferrin receptor, whichfacilitated the uptake of the VIPa-Mab conjugate through the blood-brainbarrier. Pardridge (1991); and Bickel et al. (1993) Proc. Natl. Acad.Sci. USA 90:2618-2622. Several other specific transport systems havebeen identified, these include, but are not limited to, those fortransferring insulin, or insulin-like growth factors I and II. Othersuitable, non-specific carriers include, but are not limited to,pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.Certain prodrugs have been described whereby, upon entering the centralnervous system, the drug is cleaved from the carrier to release theactive drug. U.S. Pat. No. 5,017,566.

Examples of methods of crossing the BBB include: use of vasoactivesubstances such as bradykinin or a bradykinin analog (where bradykininanalogs include, e.g., [Phe⁸ψ(CH₂—NH)Arg⁹]-bradykinin,N-acetyl-[Phe⁸ψ(CH₂—NH)Arg⁹]-bradykinin, desArg⁹-bradykinin, etc.); useof nitric oxide (NO) donor drugs (see below); localized exposure tohigh-intensity focused ultrasound; use of endogenous transport systems,including carrier-mediated transporters such as glucose and amino acidcarriers; use of liposomes loaded with nanoparticles containing anactive agent, where an example of such a nanoparticle is a polyethyleneglycol-coated hexadecylcyanoacrylate nanosphere (see, e.g., Silva (2008)BMC Neurosci. 9:S4; Brigger et al. (2002) J. Pharm. Exp. Ther. 303:928;Wong et al. (2009) Adv. Drug Del. Rev. PMID 19914319; Khalil andMainardes (2009) Curr. Drug. Del. 6:261; Modi et al. (2009) Prog.Neurobiol. 88:272; Barbu et al. (2009) Expert Opin. Drug. Del. 6:553);use of agents (e.g., Tariquidar) that inhibit P-glycoprotein at the BBB;and the like.

Suitable NO donor drugs include, e.g., organic nitrate compounds whichare nitric acid esters of mono- and polyhydric alcohols, (e.g., glyceryltrinitrate (GTN) or nitroglycerin (NTG), pentaerythrityl tetranitrate(PETN), isosorbide dinitrate (ISDN), and isosorbide 5-mononitrate(IS-5-N)), 5-nitrosothiol compounds (e.g.,S-nitroso-N-acetyl-D,L-penicillamine (SNAP), 5-nitrosoglutathione(SNOG), S-nitrosoalbumin, S-nitrosocysteine), sydnonimine compounds(e.g., molsidomine (N-ethoxycarbonyl-3-morpholino-sydnonimine),linsidomine (e.g., SIN-1; 3-morpholino-sydnonimine or3-morpholinylsydnoneimine or 5-amino-3-morpholinyl-1,2,3-oxadiazolium),and pirsidomine (CAS 936).

In other embodiments, an active agent conjugated to a targeting domainto form a chimeric therapeutic, where the targeting domain facilitatespassage of the blood-brain barrier (as described above) and/or binds oneor more molecular targets in the CNS. In some embodiments, the targetingdomain binds a target that is differentially expressed or displayed on,or in close proximity to, tissues, organs, and/or cells of interest. Insome cases, the target is preferentially distributed in a neurogenicregion of the brain, such as the dentate gyrus and/or the SVZ. Forexample, in some embodiments, an active agent is conjugated or complexedwith the fatty acid docosahexaenoic acid (DHA), which is readilytransported across the blood brain barrier and imported into cells ofthe CNS.

Combination Therapies

A TGF-β pathway inhibitor (e.g., an agent that blocks a TGF-β pathway)can be administered in monotherapy, e.g., a single TGF-β pathwayinhibitor is administered in the absence of administration of any othertherapeutic agent for the treatment of the epilepsy or theneurodegenerative disorder. Alternatively, a TGF-β pathway inhibitor(e.g., an agent that blocks a TGF-β pathway) can be administered incombination therapy with one or more additional therapeutic agents.

For example, in the treatment of epilepsy, a TGF-β pathway inhibitor canbe administered to an individual in combination therapy with one or moreadditional therapeutic agents for the treatment of epilepsy. Therapeuticagents that are suitable for administration in combination therapy witha TGF-β pathway inhibitor include, but are not limited to,Carbamazepine, Carbatrol®, Clobazam, Depakene®, Depakote®, Diastat,Dilantin®, Ethosuximide, Felbatol®, Felbamate, Frisium, Gabapentin,Gabitril®, Inovelon®, Luminal, Lyrica, Mysoline®, Neurontin®,Oxcarbazepine, Phenobarbital, Phenylek®, Phenyloin, Primidone,Rufinamde, Sabril Tegretol®, Tegretol XR®, Tiagabine, Topamax®,Topiramate, Keppra®, Keppra XR™, Klonopin, Lmaictal®, Lamotrigine,Levetiracetam, Trileptal®, Valproic Acid, Zarontin®, Zonegran®, andZonisamide.

As another example, in the treatment of Alzheimer's Disease (AD), aTGF-β pathway inhibitor can be administered to an individual incombination therapy with one or more additional therapeutic agents forthe treatment of AD. Suitable additional therapeutic agents include, butare not limited to, acetylcholinesterase inhibitors, including, but notlimited to, Aricept (donepezil), Exelon (rivastigmine), metrifonate, andtacrine (Cognex); non-steroidal anti-inflammatory agents, including, butnot limited to, ibuprofen and indomethacin; cyclooxygenase-2 (Cox2)inhibitors such as Celebrex; and monoamine oxidase inhibitors, such asSelegilene (Eldepryl or Deprenyl). Dosages for each of the above agentsare known in the art. For example, Aricept is generally administered at50 mg orally per day for 6 weeks, and, if well tolerated by theindividual, at 10 mg per day thereafter.

As another example, in the treatment of stroke, a TGF-β pathwayinhibitor can be administered to an individual in combination therapywith one or more additional therapeutic agents for the treatment ofstroke. For example, a TGF-β pathway inhibitor can be administered to anindividual in combination therapy with tissue plasminogen activator.

Subjects Suitable for Treatment

Individuals who are suitable for treatment with a TGF-β inhibitorpathway include individuals who have been diagnosed with epilepsy;individuals who have suffered a stroke; individuals who have sufferedtraumatic head injury; individuals who have suffered brain infection(i.e. viral or bacterial encephalitis) and individuals having aneurodegenerative disorder or neurological symptoms due to diseases ofsmall blood vessels (vasculitis, diabetes mellitus).

Subjects suitable for treatment with a subject method includeindividuals having one or more of the following disorders: epilepsy,traumatic brain injury, stroke, brain infection (i.e. viral or bacterialencephalitis) and neurodegenerative diseases (including that resultedfrom a small vessel disease, e.g. diabetes mellitus). A subject methodis suitable for treating epilepsy (including posttraumatic epilepsy),Parkinson's disease, psychosis, migraine, cerebral ischemic, Alzheimer'sdisease and other degenerative diseases such as Huntington's chorea,schizophrenia, obsessive compulsive disorders (OCD), neurologicaldeficits associated with acquired immunodeficiency syndrome, traumaticbrain injury, inappropriate neuronal activity resulting inneurodysthesias in diseases such as diabetes, multiple sclerosis (MS)and motor neuron disease, ataxias, muscular rigidity (spasticity),temporomandibular joint dysfunction, and amyotrophic lateral sclerosis(ALS).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 TGF-β Signaling in Epileptogenesis Experimental Procedures

In vivo preparation: All experimental procedures were approved by theethical committees dealing with experiments on animals at CharitéUniversity Medicine, Berlin and Ben-Gurion University of the Negev,Beer-Sheva. The in vivo experiments were performed as previouslydescribed (Ivens et al., 2007; Seiffert et al., 2004). Adult male Wistarrats were treated with artificial cerebrospinal fluid (aCSF, compositionin mM: 129 NaCl, 21 NaHCO3, 1.25 NaH2PO4, 1.8 MgSO4, 1.6 CaCl2, 3 KCl,10 glucose) supplemented with deoxycholic acid (DOC, 2 mM,Sigma-Aldrich, Steinheim, Germany), bovine serum albumin (BSA, 0.1 mM,Merck, Darmstadt, Germany) corresponding to 25% of serum albuminconcentration, or with TGF-β1 (10 ng/ml, Peprotech, Rocky Hill, N.J.).Sham-operated animals (perfused with aCSF) served as controls. Only ratswith no apparent injury to the cortical surface or bleeding fromcortical vessels (as seen under the surgical microscope) at the end ofthe procedure were used. Animals were sacrificed 7/8, 24, or 48 hoursfollowing treatment. A second set of animals including sham-operatedcontrols and animals treated with BSA or BSA plus TGF-βR blockers(TGF-βRII antibody, 50 μg/ml, Santa Cruz Biotechnology, Santa Cruz,Calif.; SB431542, 100 μM, Tocris, Bristol, UK) were sacrificed 24 hoursfollowing treatment. For Smad2-P immunodetection, animals were treatedwith 0.2 mM BSA and sacrificed 46-50 hours following treatment.

In vitro slice preparation: Brain slices for the in vitro experimentswere prepared by means of standard techniques (Ivens et al., 2007;Pavlovsky et al., 2003; Seiffert et al., 2004). Slices were transferredto a recording chamber where they were incubated in aCSF containing BSA(6.7 mg/ml), TGF-β1 (10 ng/ml) or artificial serum (aSerum, compositionbased on aCSF with the following changes, composition in mM: 0.8 MgSO4,1.3 CaCl2, 5.7 KCl, 1 L-glutamine, 0.1 albumin). To block the activityof TGF-β1, slices were incubated in aCSF containing SB431542 (10 μM)before the addition of TGF-β1 (10 ng/ml). To block TGF-βRs, slices wereincubated in aCSF containing SB431542 (10 μM) and TGF-βRII antibody (10μg/ml) for 30 min followed by incubation in BSA in the presence ofTGF-βR blockers. For detection of epileptiform activity, fieldpotentials were recorded >4 h following incubation in cortical layer IVusing extracellular glass microelectrodes (˜3MΩ) in response to bipolarstimulation at the border of white and grey matter.

Albumin and TGF-βRII Co-Immunoprecipitation: To prepare corticallysates, brains were isolated from adult Wistar rats, dissected in coldsaline solution and lysed in RIPA buffer. BSA (3 μg) was added tolysates to approximately match the amount of precipitating anti-albuminantibodies. Immunoprecipitation was performed using the Catch andRelease® v2.0 Reversible Immunoprecipitation System (Upstate,Charlottesville, Va.) with the following modifications to the standardprotocol: the starting amount of protein was increased to 1,500 μg andthe incubation time with precipitating antibodies was increased to 90minutes. Lysate samples (positive or negative for albumin) wereimmunoprecipitated with an anti-TGF-βRII antibody (Upstate) or ananti-albumin antibody (Biogenesis, Poole, UK).

The immunoprecipitated samples were separated with SDS-PAGE andtransferred onto a nitrocellulose membrane. The membrane was stainedwith Ponceau S stain to confirm that the IP procedure was successful. Itwas then destained, and blocked with 5% BSA in standard TBS-T bufferovernight at 4° C. TGF-βRII was detected with a rabbit anti-TGF-βRIIantibody (Upstate) and an AP-conjugated donkey anti-rabbit IgG secondaryantibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.).Chemiluminescent detection was done using Lumi-Phos WB ChemiluminescentSubstrate (Pierce, Rockfort, Ill.) and standard X-ray film according tothe manufacturer's instructions.

Smad2-P Western Blot Analysis: Cortical lysate samples fromsham-operated controls and animals treated with BSA were separated bySDS-PAGE and transferred onto a nitrocellulose membrane. The membranewas blocked with 5% nonfat milk overnight at 4° C., incubated with arabbit polyclonal antibody against phospho-Smad2 (Millipore Corporation,Bedford, Mass.) for 48 hr at 4° C., and incubated with aperoxidase-conjugated goat anti-rabbit IgG secondary antibody (JacksonImmunoResearch Laboratories) for 2 hr at room temperature.

Microarrays: Total RNA was isolated using the TRIzol reagent(Invitrogen, Carlsbad, Calif.) and prepared using the AffymetrixGeneChip one-cycle target labeling kit (Affymetrix, Santa Clara,Calif.). Biotinylated cRNA was fragmented and hybridized to the GeneChipRat Genome 230 2.0 Array according to company protocols (AffymetrixTechnical Manual). Normalization of the array data was done using GCRMA(GC Robust Multi-Array Average) analysis. Functional annotation analysiswas performed with the program Database for Annotation, Visualization,and Integrated Discovery (DAVID) 2008 (Dennis et al., 2003)(http:(double forward slash)david(dot)abcc(dot)ncifcrf(dot)gov). TheGenMAPP 2.0 program (Salomonis et al., 2007) (http://www.genmapp.org/)was used to visualize genes involved in TGF-β signaling. For the timecourse analysis one array was run for each treatment (DOC, BSA, TGF-β1)for the following time points: 7/8, 24, and 48 hr. In addition a samplefrom a sham treated animal (24 hr) was run and used to normalize theother arrays. Pairwise Pearson correlation coefficients for the threetreatments were determined with Excel (Microsoft Corp., Richmod, Wash.).Hierarchical clustering was performed with Gene Cluster and displayedwith TreeView software (Eisen et al., 1998). Arrays were then run forthe second set of animals sacrificed 24 hr following treatment (Sham,n=2; BSA, n=3; BSA+TGF-βR blockers, n=4). Significance analysis ofmicroarrays (SAM) was performed with a false discovery rate (FDR)threshold of 9.2%. A 1.5 fold change cutoff was also used to filter thislist. Genes which demonstrated a significant change in expressionfollowing albumin treatment and a log 2 ratio difference>0.5 between thetwo treatments were considered part of the attenuated response. Genesdemonstrating a significant change in expression following albumin andalbumin plus blocker treatments were considered part of the unattenuatedresponse. All microarray data are available at the GEO website(http://www.ncbi.nlm.nih.gov/geo) under accession number GSE12304.

Real-time RT-PCR: mRNA expression levels were determined by quantitativereverse transcriptase-PCR by real-time kinetic analysis with an iQ5detection system (Bio-Rad, Hercules, Calif.). Real-time PCR data wereanalyzed using the PCR Miner program (Zhao and Fernald, 2005). 18S mRNAlevels were used as internal controls for variations in samplepreparation. Primer sequences are provided in supplementary methods.

Statistical Analyses: For the electrophysiological data, differencesbetween treated and control slices were determined by the Mann-Whitney Utest for two independent samples or the chi-square test using SPSS13.0(SPSS Inc., Chicago, Ill.). Linear regression analysis for themicroarray data was performed with Graphpad Prism (GraphPad Software,Inc., San Diego, Calif.). PCR data were analyzed with an unpairedStudent's t test (p<0.05 was considered significant) in Excel(Microsoft) or with the relative expression software tool (REST)(Pfaffl, 2001). REST determines significance of the group ratio resultswith a randomization test. p<0.05 was taken as the level of statisticalsignificance.

Results TGF-β Signaling is Sufficient to Induce Epileptiform Activity

To assess the hypothesis that activation of the TGF-β signaling pathwayis the mechanism underlying albumin-induced epileptogenesis, weactivated this pathway directly by incubating neocortical slices withTGF-β1 (10 ng/mL) in artificial cerebrospinal fluid (aCSF) and performedelectrophysiological recordings. These recordings were compared to thoseof slices treated with a solution containing serum levels ofelectrolytes and 0.1 mM albumin (aSERUM, previously shown to induceepileptogenesis; Ivens et al., 2007), albumin in aCSF, or aCSF(control). Spontaneous, prolonged and hypersynchronous interictal-likeactivity was observed in slices treated with aSERUM for 6-10 hours (n=6out of 9 slices, 3 animals) but never in aCSF treated slices (FIG. 1 b).When albumin was added to the control aCSF solution, epileptiformactivity was recorded in response to stimulation of the white matter(n=8 out of 12 slices, 6 animals). Importantly, TGF-β1 treatment wassufficient to recapitulate epileptiform activity similar to that seenfollowing treatment with aSERUM and albumin in aCSF (n=5 out of 5slices, 4 animals; n=7 out of 9 slices, 3 animals; and n=8 out of 12slices, 6 animals, respectively). In all three cases, the evokedepileptiform activity, was all-or-none in nature, paroxysmal andprolonged, similar to that seen following BBB opening with bile salts(Ivens et al., 2007; Seiffert et al., 2004) and typical to that observedin acute models of epilepsy (Gutnick et al., 1982). No activity was seenin the aCSF control treated slices.

To further confirm that the TGF-β1 induced epileptiform activity wasdependent on the TGF-βR mediated pathway, we performed additional trialsof the above experiments in the presence of two TGF-βR blockers(SB431542, the TGF-βRI kinase activity inhibitor and TGF-βRII antibody).TGF-βR blockers prevented epileptiform activity induced by TGF-β1 oralbumin (FIG. 1 c). The measured integral of the field potential(albumin: 117.2±35.4 mV*ms; TGF-β1: 84.1±20.1 mV*ms) was significantlylower in slices treated with albumin or TGF-β1 in the presence of TGF-βRblockers (albumin and blockers: 23.7±6.9 mV*ms, n=20 slices, 4 animals,p=0.001; TGF-β1 and blockers: 12.5±3.9 mV*ms, n=8 slices, 4 animals,p=0.005) (FIG. 1 d).

Albumin Binds TGF-βRs and Activates the TGF-β Pathway

To determine whether albumin binds to TGF-β receptors,co-immunoprecipitation using antibodies against albumin or TGF-βRII wasperformed on cortical lysate samples treated with albumin. An expectedband corresponding to TGF-βRII was detected in samplesimmunoprecipitated with the TGF-βRII antibody. More importantly, thisband was also detected in samples pre-incubated with albumin whenimmunoprecipitated with the albumin antibody and probed for TGF-βRII(FIG. 2 a). These results reveal a direct interaction between albuminand TGF-βRII. In the canonical TGF-β signaling pathway, Smad2 and/or 3are phosphorylated following TGF-β receptor activation and form acomplex with Smad4, which then translocates into the nucleus andactivates transcription (Shi and Massague, 2003). To investigate whetheralbumin activates downstream components of the TGF-β pathway, Smad2phosphorylation levels in cortical lysates were assessed by Westernblot, revealing an increase in Smad2 phosphorylation in animals exposedto albumin for 48 hours as compared to sham-operated controls (FIG. 2b).

Similar Transcriptional Profiles Follow BBB Opening, Albumin and TGF-β1Treatments

BBB opening or exposure to albumin in vivo (Ivens et al., 2007), as wellas in vitro exposure of neocortical slices to albumin or TGF-β1 (FIG. 1c) all result in the gradual development of hypersynchronous neuronalepileptiform activity. The delayed appearance of abnormal activity (5-7hours in vitro and >4 days in vivo, data not shown (Ivens et al., 2007))suggests a transcription-mediated mechanism. In search of a commonpathway and transcriptional activation pattern that underlieepileptogenesis following BBB opening, we performed transcriptomeanalysis using Affymetrix rat microarrays. RNA was extracted fromcortical regions of rats treated with sodium deoxycholate (DOC, toinduce BBB opening), albumin or TGF-β1 for various durations (7/8, 24,48 hr). Control RNA was extracted from cortical regions excised fromsham-operated animals. Hierarchical clustering analysis of these arraysshowed that overall the three treatments resulted in strikingly similargene expression changes, as arrays representing similar time pointsclustered together regardless of the treatment (FIG. 3 a). Thesesimilarities are exemplified in FIG. 3 b, which shows a high correlationbetween the expression profiles for the albumin and TGF-β1 treatments at24 hours (r²=0.75, p<0.0001).

To identify biological themes common to the three treatments, the genelist was filtered to include genes showing at least a 1.5 fold change inexpression and a Pearson correlation coefficient≧0.95 for pair-wisecomparisons between all treatments (see methods). Hierarchicalclustering was performed and the main clusters were used for geneontology (GO) analysis with DAVID (Database for Annotation,Visualization, and Integrated Discovery; Dennis et al., 2003). Molecularfunction and biological process GO terms with a p-value<0.05 containingat least three genes were considered significant. This analysis revealsmajor gene expression trends that occur in response to all threeepileptogenic treatments (FIG. 3 c). Early responses include genesinvolved in general stress-related cellular, metabolic and intracellularsignaling pathways; early responses persisting to later time pointsinclude inflammatory processes as well as genes involved in induction ofcell cycle, differentiation, proliferation, and apoptosis; responses atmiddle to late time points include repression of synaptic transmissionand ion transport genes (FIG. 3 c).

Gene Level Expression Profiles

Selected GO term groups were chosen for further analysis of individualgene expression profiles (FIG. 4). The most dramatic change observed inall treatments across all time points was the early and persistentupregulation of genes associated with immune response activation,including inflammatory NF-kappa B pathway related genes, cytokines andchemokines (FIG. 4 a), and complement pathway genes (FIG. 4 b). Asignificant neuronal response was prominent in the middle-late timepoints and included downregulation of genes associated with GABAergic(inhibitory) neurotransmission (FIG. 4 c) and modulation of genesassociated with glutamatergic (excitatory) neurotransmission again (FIG.4 d). Furthermore, a variety of voltage gated ion channels includingcalcium, sodium, chloride, and potassium channels were affected by allthree epileptogenic treatments (FIG. 4 e), including a noteworthydownregulation of voltage gated (Kv7.3 and Kv8.1) and inward rectifying(Kir3.1) potassium channels. We also found significant modulation ofglial-specific genes beginning in the early time point (FIG. 4 f): thecytoskeletal proteins GFAP and vimentin (Vim), and several calciumbinding proteins (S100a6, S100a10, s100a11) were all upregulated whilegap junction connexins 30 and 43 (Cx30 and Cx43) and the inwardrectifying potassium channel Kir4.1 were downregulated. Microarray-basedgene expression measurements for selected genes were further verifiedusing quantitative real-time PCR. Expression patterns were similaralthough the magnitude of the fold changes sometimes differed.

TGF-β Pathway Activation is Required for Transcriptional Changes

Given the high correlation between expression profiles following thethree epileptogenic treatments, combined with the biochemical evidencethat albumin binds to TGF-β receptors and the physiological evidencethat TGF-β treatment induces epileptogenesis, we assessed the extent towhich each treatment activates transcription of genes known to beassociated with the TGF-β signaling pathway using GenMAPP (Salomonis etal., 2007). Genes which showed at least a 1.5 fold change in expressionfollowing albumin or TGF-β1 treatment, are highlighted in FIG. 5,demonstrating that 86% of genes modulated by TGF-β1 treatment are alsomodulated following albumin treatment.

The above evidence indicates that TGF-β signaling is a key mediator ofalbumin-induced epileptogenesis. To determine if the globaltranscriptional response seen following albumin treatment is dependenton activation of the TGF-β signaling pathway, we performed an additionalset of microarray expression profiles using rats treated with albumin inthe absence (n=3) or presence of TGF-βRI and II blockers (n=4, TGF-βRIkinase activity inhibitor SB431542 and anti-TGF-βRII antibody) andsacrificed 24 hours following treatment. Although some changes in geneexpression resulting from albumin treatment were still present followingthe blocker treatment, the majority of these changes were absent orattenuated following TGF-β pathway blocker treatment (FIG. 6 a),confirming dependence of the albumin-induced transcriptional response onTGF-β signaling.

Gene ontology analysis was then used to reveal which biologicalprocesses were blocked following TGF-β pathway blocker treatment (FIG. 6a). Genes in the TGF-β signaling GO term demonstrated a dramaticsuppression of the albumin-induced expression changes in the presence ofTGF-β signaling blockers (FIG. 6 b). In addition, TGF-β pathway blockertreatment prevented the albumin-induced modulation of genes involved inneuronal processes, immune response, and ion and cellular transport(FIG. 6 b). Several prominent signaling pathways including the NF-kappaBcascade, Jak-Stat cascade, and MAPKKK cascade were upregulated followingalbumin treatment, but did not show a similar upregulation followingalbumin treatment in the presence of TGF-β pathway blockers.Quantitative real-time PCR was also performed with these samples toconfirm the microarray results (FIG. 6 c). Indeed, TGF-β pathway blockertreatment completely blocked expression changes following albuminexposure for Stat3 and Glt-1 and partially blocked changes for Cx43 andGFAP.

FIGS. 1-6

FIG. 1: Epileptiform activity and TGF-β pathway activation are inducedby serum albumin. (A) Photograph of a brain slice displaying electrodepositioning. (B) Extracellular recordings showing spontaneousinterictal-like epileptiform activity following treatment withartificial serum containing albumin (aSERUM). (C) Evoked responses fromslices treated with aCSF, albumin or TGF-β1 in aCSF, or albumin inartificial serum (aSERUM). (D) Albumin and TGF-βRIIimmunoprecipitations. Samples treated or untreated with serum albuminwere co-immunoprecipitated with antibodies directed against albumin orthe TGF-βRII receptor. All samples were then probed with ananti-TGF-βRII antibody. The band at 50 kDa is the heavy chain of theprecipitating antibody. (E) Western blot analysis of Smad2-P followingalbumin treatment.

FIG. 2: Genome wide transcriptional analysis following epileptogenictreatments. (A) Hierarchical clustering of arrays corresponding to 7/8,24 and 48 hours following DOC, albumin and TGF-β1 treatments. Note howarrays cluster together for each time point across all treatments. (B)Linear regression analysis between TGF-β1 and albumin treatments at 24hours. Only genes with a fold change equal to or greater than 1.5 forthe TGF-β1 treatment were included. (C) Hierarchical cluster analysis ofgenes showing correlation (>0.95) between all treatments. Selectedclusters were annotated with DAVID to reveal biological themes common toall treatments. Color bar indicates range of log₂ ratios.

FIG. 3: Gene ontology (GO) annotation analysis. Log₂ ratios for selectedgenes from GO annotation analysis involved in (A) inflammation, (B)complement activation, (C) GABAergic transmission, (D) glutamatergictransmission, (E) voltage gated ion channels, and (F) Astrocytic-relatedgenes. Numbers below data points correspond to the various treatments(7/8, 24, and 48 hours).

FIG. 4: qRT-PCR gene expression analysis. Time course analysis forselected genes following treatment with DOC, albumin or TGF-β1 at 7/8,24 or 48 hr. Data are expressed as fold changes relative to sham treatedcontrols. Significance of changes was assessed for the 24 hour timepoint (shown in insets for albumin (n=3) and TGF-β1 (n=3) treatments;error bars indicate s.e.m. asterisks indicate p<0.05).

FIG. 5: Albumin alters TGF-β pathway gene expression. TGF-β pathway mapgenerated with GENMAPP software illustrating significant changes (>1.5or <−1.5 fold change) in gene expression following albumin treatment incomparison to TGF-β1 treatment. Yellow labeled genes represent genes upor downregulated following albumin treatment, blue labeled genesrepresent genes up or downregulated following TGF-β1 treatment, andgreen labeled genes represent genes up or downregulated following bothtreatments. Gene pathway map created by Nurit Gal and Manny Ramirez,Copyright 2002, Gladstone Institute.

FIG. 6: Blocking TGF-β signaling prevents albumin-induced geneexpression and epileptiform activity. (A) Genomic expression analysisfollowing treatment with albumin or albumin plus TGF-β receptorblockers. Gene ontology analysis was performed with DAVID for genesshowing an attenuated [(albumin log 2ratio)−(albumin+blocker log2ratio)>0.5] or unattenuated response following treatment with albuminplus TGF-β receptor blockers in comparison to albumin treatment. (B)Fold changes for specific genes from GO analysis. (C) qPCR analysis forselected genes following albumin (n=3) or albumin plus TGF-β receptorblockers (n=4). (D and E) TGF-β receptor blockers prevent epileptiformactivity induced by albumin or TGF-β1 treatment. Comparison of meanevent integral in the 50-500 ms time range shows a significant increasein the field potential integral in the albumin and TGF-β1 treated slicesbut not in slices treated with TGF-β receptor blockers. Error barsindicate s.e.m. Asterisks indicate p<0.05.

Example 2 Effect of Losartan Potassium Treatment

Wistar male rats (160-190 g) were deeply anesthetized with Ketamin (311mg/Kg body weight) and Xylazine (11 mg/Kg body weight) and placed in astereotactic frame. Sagittal incision was made and a rectangular bonewindow was drilled over the sensory-motor cortex. The dura was removedand the underlying brain perfused for 40 minutes with aCSF forsham-operated animals (Group A; n=2); bovine serum albumin (BSA)dissolved in aCSF for treated animals (Group B; n=4); or a mixture ofBSA and losartan potassium dissolved in aCSF for a second group oftreated animals (Group C; n=4). BSA concentration was 0.2 mM (>98% inagarose gel electrophoresis; Sigma) and the concentration of Losartanpotassium was 10 μM. The composition of the aCSF was (in mM): 129 NaCl,21 NaHCO₃, 1.25 NaH₂PO₄, 1.8 MgSO₄, 1.6 CaCl₂, 3 KCl and 10 glucose.After perfusion, the bone window was carefully closed and the skin wassutured. Only rats with no apparent injury to the cortical surface ofbleeding from cortical vessels, as seen under the surgical microscope atthe end of the procedure, were included in this study.

Animals were sacrificed 46-50 hours after surgery, the brain was removedfrom each animal, and the treated area was dissected. Western blotanalysis was used to detect the levels of phosphorylated Smad2 and Smad3proteins. The results are shown in FIG. 7.

Changes in mRNA expression levels were determined for animals treatedwith BSA or BSA in addition to losartan for 24 hours. Quantitativereverse transcriptase-PCR by real-time kinetic analysis was performedwith an iQ5 detection system (Bio-Rad, Hercules, Calif.). Real-time PCRdata were analyzed using the PCR Miner program (Leonoudakis et al.,2008) and 18S mRNA levels were used as internal controls for variationsin sample preparation. The results are shown in FIG. 8.

Brain activity was recorded from awake, behaving rats during the“epileptogenic” time window using implanted electrodes positioned on thesurface of the cerebral cortex (Data Science International, USA). TheEEG data is depicted in FIG. 9A-D. Signal harmony (A) and “complexity”(fractal dimension-B) were computed, and shown to increase and decrease,respectively, during albumin-induced epileptogenesis (BSA, big brokenline)—indicating a gradual increase in network synchronicity. Using a“home made” automatic detection of “seizure like events” (SLEs), a sharpincrease in their number one week following treatment was observed. Asingle, focal application of Losartan together with BSA reversed thealbumin-induced EEG changes observed during epileptogenesis (A-B, smallbroken line) to those similar to sham-controls (unbroken line).Furthermore, the number of seizure like events (SLEs) was significantlysmaller in losartan-treated rats (n=7; FIG. 9C), compared to BSA-treatedones (n=8, FIG. 9D).

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Example 3 Astrocyte Dysfunction in Epileptogenesis Materials and Methods

Animals were housed and handled according to the directives of theinternationally accredited Animal Care and Use Committees (IACUC) atCharité University Medicine, Berlin, and Ben-Gurion University of theNegev, Beer-Sheva. All experimental procedures were approved by theethical committees supervising experiments on animals at CharitéUniversity Medicine (in-vivo approval no.: G0104/05, in-vitro: T0228/04)and Ben-Gurion University of the Negev (approval no.: BGU-R-71-2006).

In-vivo experiments. The in-vivo experiments were performed aspreviously described in Seiffert et al. (2004). In brief, adult maleWistar rats (120-250 g) were anesthetized using ketamine and xylazineand placed in a stereotactic frame. A 4-mm diameter bone window wasdrilled over the somatosensory cortex, the dura was opened and theunderlying cortex was perfused with artificial cerebrospinal fluid(ACSF). For the “treated” rats group, the BBB-disrupting agentdeoxycholic acid sodium salt (DOC, 2 mM, Sigma-Aldrich, Steinheim,Germany) or bovine serum albumin (0.1 mM, >98% in agarose cellelectrophoresis; catalogue no. A7906, Sigma Aldrich, Steinheim, Germany)was added to the ACSF. Albumin concentrations corresponded to 25% of thenormal serum concentration [determined to be 0.4 mM for 10 rats, seealso Geursen and Grigor (1987); final osmolarity of 303-305 mOsmol/l].For the sham-operated control group, the cortex was perfused with ACSF.The composition of the ACSF was (in mM): 129 NaCl, 21 NaHCO₃, 1.25NaH₂PO₄, 1.8 MgSO₄, 1.6 CaCl₂, 3KCl, and 10 glucose. Rats weresacrificed at 7-8, 24, or 48 h following treatment, before the onset ofepileptiform activity (>4 days, see Seiffert et al., 2004).

Microarrays. Total RNA from animals treated with DOC or with albumin wasisolated from the somatosensory cortex, directly under the craniotomyarea, using the TRIzol® reagent (Invitrogen, Carlsbad, Calif.), andprepared using the Affymetrix GeneChip one-cycle target labeling kit(Affymetrix, Santa Clara, Calif.). Biotinylated cRNA was then fragmentedand hybridized to the GeneChip Rat Genome 230 2.0 Array according tomanufacturer's protocols (Affymetrix Technical Manual). The array datawas normalized by using GCRMA (GC Robust Multi-Array Average) or RMA(Robust Multi-Array Average) analysis. One array was run for eachtreatment (DOC and albumin) and for every contralateral hemisphere forthe following time points: 7/8, 24, and 48 h. The data from asham-treated animal (24 h) was used to normalize the other arrays. Toidentify genes involved in astrocytic functions, GeneCards(http://www.genecards.org), querying for “astrocyte”, was used. Forcomparison of the relative changes in the expression of astrocytic vs.neuronal genes, gene sets of astrocytic and neuronal enriched genes(expressed by S100β+ and S100β−/PDGFRα−/MOG-cells, respectively) wereused (Cahoy et al. J Neurosci 28:264-278, 2008). Cluster analysis wasperformed with MATLAB by assessing the expression relationship as theEuclidean distance in N-dimensional space between measurements (Ndenotes number of gene transcripts). Arrays were then clusteredaccording to distance data, by using the Unweighted Pair Group Methodwith Arithmetic mean method (UPGMA, Gronau and Moran, 2007).

In-vitro astrocytic and neuronal culture preparations. Primary neuronalcortical cultures were prepared from embryonic day 18 rats as reportedpreviously (Kaufer et al., 2004). Briefly, cells were dissociated with apapain solution for 20 min at 37° C. After the removal of the papainsolution, the tissue was resuspended in growth medium [MEM with Earle'ssalts containing 2.5% B27 supplement, 0.1% mito serum extender, 5% fetalbovine serum (FBS), 20 mM glucose, and 5 mM L-glutamine] and dissociatedby mechanical trituration. The cells were plated, and after 4 h in vitrothe cell culture medium was replaced with neurobasal medium supplementedwith 2% B27 supplement and 0.5 mM GlutaMAX™. The cells were maintainedin 5% CO₂ at 37° C. After 7 days in vitro, cytosine arabinofuranoside(AraC) (10 μM) was added to the cultures. After 10 days in vitro, thecells were incubated with 0.4 mM albumin for 24 h at 37° C. Forastrocytic cultures, astrocytes were isolated from the cerebral corticesof PO rat pups. Cells were dissociated with papain and mechanicaltrituration. The cells were cultured in high-glucose Dulbecco's modifiedeagle medium supplemented with 10% FBS and 1% penicillin/streptomycin at37° C. and in 5% CO2 (medium was replaced every 3-4 days). After 10 daysin vitro, the culture medium was replaced with serum-free high-glucoseDMEM (containing 1% penicillin/streptomycin) for 18 h. The cells werethen incubated in serum-free medium containing 0.4 mM albumin for 24 hat 37° C. For immunostainings cells were washed with phosphate bufferedsaline (PBS) and fixed in 4% paraformaldehyde for 15 min. The cells werepermeabilized with 0.2% Triton X-100 in PBS for 5 min and washed in PBS.They were then incubated with 5% normal donkey serum in PBS for one hourat room temperature followed by overnight incubation at 4° C. witheither mouse anti-NeuN (1:1000; Chemicon, Temecula, Calif.) or mouseanti-GFAP (1:1000; Cell Signaling Technology, Beverly, Mass.). The cellswere washed in PBS, incubated with donkey anti-mouse Cy3 (1:1000;Jackson ImmunoResearch, West Grove, Pa.) for 1 hour at room temperature,and then counterstained with DAPI.

Real-time polymerase chain reaction. Total RNA was isolated from thesomatosensory cortices of animals treated with DOC or albumin (24 htreatment; n=3) or from primary cultures (astrocytic and neuronal, n=3independent experiments). Expression levels were determined by real-timereverse transcriptase-PCR (RT-PCR) with an iQ5 detection system(Bio-Rad, Hercules, Calif.) using gene-specific primer pairs. RT-PCRdata were analyzed using the PCR Miner program (Zhao and Fernald, 2005),and fold changes in gene expression were represented relative tosham-operated controls (in-vivo samples) or serum-deprived controls(in-vitro samples). Ribosomal 18S RNA (18S rRNA) was used as an internalcontrol for variations in sample preparation. For samples from in-vivotreatments, RT-PCR was performed with the iScript one-step RT-PCR kit(Bio-Rad). Control RT-PCR reactions were performed without reversetranscriptase to verify amplification of genomic DNA. For in-vitrosamples, DNase treatment was applied, followed by first-strand cDNAsynthesis (iScript cDNA Synthesis kit, Bio-Rad). PCR reactions werecarried out with iQ SYBR Green Supermix (Bio-Rad). Primer specificitywas verified by melt curve analysis. The amplification cycles for 18S,Gja1, GS, SLC1A2, SLC1A3 (GLAST) and Kcnj10 consisted of 40 cycles of 10s at 95° C., 30 s at 55° C., and 30 s at 72° C. The amplification cyclesfor Gjb2 and Gjb6 consisted of 40 cycles of 10 s at 95° C., 30 s at 60°C., and 30 s at 72° C.

Primer sequences (forward, reverse) were as follows: 18S rRNA (GenBankaccession number M11188.1, 5′-CCATCCAATCGGTAGTAGCG-3′ (SEQ ID NO:1), 5′GTAACCCGTTGAACCCCATT-3′ (SEQ ID NO:2)); SLC1A3 (GenBank accession numberNM_(—)019225.1; 5′-GAGGCCATGGAGACTCTGAC-3′ (SEQ ID NO:3),5′-CGAAGCACATGGAGAAGACA-3′ (SEQ ID NO:4)); GS (GenBank accession numberNM_(—)017073.3; 5′-AGCGACATGTACCTCCATCC-3′ (SEQ ID NO:5), 5″TACAGCTGTGCCTCAGGTTG-3′ (SEQ ID NO:6)); Kcnj10 (GenBank accession numberX83585.1; 5′-GAGACGACGCAGACAGAGAG-3′ (SEQ ID NO:7),5′CCACTGCATGTCAATGAAGG-3′ (SEQ ID NO:8)); Gjb2 (GenBank accession numberNM_(—)001004099.1; 5′-GGTTTGTGATGTGAGCATGG-3′ (SEQ ID NO:9),5′-CTCAGCACACCAAGGATGAA-3′(SEQ ID NO:10)); Gjb6 (GenBank accessionnumber NM_(—)053388.1; 5′-GCCAAGATGAGTCACAGCAA-3′ (SEQ ID NO:11),5′-TCAGAGCTGGATCACAATCG-3′ (SEQ ID NO:12)); Gjal (GenBank accessionnumber NM_(—)012567.2; 5′-TCCTTGGTGTCTCTCGCTTT-3′ (SEQ ID NO:13),5′-TTTGGAGATCCGCAGTCTTT-3′ (SEQ ID NO:14)); SLC1A2 (GenBank accessionnumber NM_(—)017215.2; 5′-GGTCAATGTAGTGGGCGATT-3′ (SEQ ID NO:15),5′-GGACTGCGTCTTGGTCATTT-3′ (SEQ ID NO:16)).

In-vitro electrophysiological recordings. For electrophysiologicalexperiments, rats were deeply anesthetized with isoflurane and thendecapitated. Brains were quickly removed, and transversehippocampal-cortical slices (400 μm thick) were prepared using avibratome (Campden Instruments, Loughborough, UK). Slices weremaintained in a humidified, carbogenated (5% CO₂ and 95% O₂) gasatmosphere at 36±1° C. and perfused with ACSF in a standard interfacechamber (Seiffert et al., 2004; Ivens et al., 2007). To mimic thealtered ionic environment during BBB disruption, recordings wereacquired in a serum-adapted electrolyte solution (sACSF; see Seiffert etal. 2004). sACSF was similar in composition to the ACSF except fordifferent concentrations of MgSO₄ (0.8 mM), CaCl₂ (1.3 mM), KCl (5.7 mM)and glutamine (1 mM). “Treated” slices were incubated with sACSFcontaining 0.1 mM bovine serum albumin for 2 h before transfer to theperfusion chamber.

Electrophysiological recordings were obtained 6-10 h following perfusionwith sACSF. Control slices were treated similarly, using sACSF withoutalbumin. For extracellular recordings, glass microelectrodes (˜3 MΩ, 154mM NaCl) were positioned in layer 4 of the neocortex. Slices werestimulated with brief (100 μs) pulses, by using bipolar stimulationelectrodes placed at the border between white and gray matter in thesame cortical column. Trains of 50 stimuli were applied at 2, 5, 10, 20,50 and 100 Hz, at 2.5× threshold stimulation intensity. Signals wereamplified (SEC-10L; NPI Electronics, Tamm, Germany), filtered at 2 kHz,displayed on an oscilloscope, digitized on-line (CED-1401 micro;Cambridge Electronics Design, Cambridge, UK) and stored for off-lineanalysis. Extracellular potassium concentrations ([K+]_(o)) weremeasured with ion-sensitive microelectrodes (ISMEs; Lux and Neher, 1973;Jauch et al., 2002).

In vitro intracellular recordings were obtained from pyramidal neurons(layer 2-3) 23-28 h following the in vivo treatment with albumin or fromcontrol rats. Currents were recorded using the whole cell patchconfiguration, as described previously (Pavlovsky et al., 2003). Inbrief, glass pipettes were pulled from capillaries using a verticalpuller (Narashige, Greenvale, N.Y.) and filled with a solutioncomprising (in mM): 150CsCl, 1MgCl₂, 10 HEPES, 4Na₂ATP, 0.1CaCl₂, and1.1 mM EGTA, pH adjusted to 7.2 with a final osmolarity of 290-310 mOsm.Cells were visualized using infrared differential interference phasecontrast video-microscopy. Recordings were performed using AxoPatch 700B(Axon Instruments, Foster City, Calif.), digitized at 10 kHz andrecorded using pClamp 9.2 (Axon Instruments, Foster City, Calif.). Patchpipette's resistance was 4-5 Ma Series resistance was not electronicallycompensated; however, cells in which series resistance varied by morethan 25% were excluded from the analysis. Stimulation protocols werestarted at least 5 min following impalement to allow intracellulardialysis with the pipette solution. Excitatory post-synaptic currents(EPSCs) were evoked—using a bipolar stimulating electrode positioned<200 μm from the recorded cell—at 75% of the intensity producing maximalEPSCs. N-methyl-D-aspartic acid (NMDA) currents were recorded in thepresence of blockers of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA)/kainate (i.e., 301 μM CNQX) and of gamma-aminobutyric acid A(GABA_(A)) receptors (i.e., 10 μM bicuculine methiodide). Cells werevoltage clamped to +40 mV to alleviate NMDA receptor blockade andinactivate fast Na⁺ currents. In some experiments, dihydrokainic acid(DHK, Tocris, Bristol, UK), 100 μM, was added to the extracellularsolution to selectively block the astrocyte specific glutamatetransporter, SLC1A2 (see Arriza et al., 1994).

Computer simulations. A computer model was implemented using the NEURONmodeling environment (Hines and Carnevale, 1997) with 20-μs time steps.The model consisted of a multi-compartment isolated cell, simulating alayer 2/3 cortical neuron, using only passive membrane properties.Geometric parameters and spatial relationships of the 74 compartmentswere modeled after Traub and colleagues (2003). Resting membranepotential was set at −65 mV (determined by Na⁺ and K⁺ conductance);membrane capacitance C_(m) was 0.9 μF/cm²; and the cytoplasmicresistance was set at 250 Ω/cm². Simulated excitatory inputs consistedof eight synapses on apical dendrites (located 1368 μm from the soma),contributing currents with AMPA and NMDA kinetics modeled after Saftenku(2005) and Kampa et al. (2004), respectively. AMPA to NMDA maximalcurrent ratios were set at 1 (Myme et al., 2003). Synaptic currents weretriggered by a surge of ‘glutamate’, decaying with first-order kinetics(baseline time constant=1.2 ms). Down-regulation of uptake mechanismswas simulated by changing the time constant of the decay function,similar to the effect of the application ofDL-threo-β-benzyloxyaspartate (DL-TBOA, Diamond, 2005). To investigatethe effects of altered [K⁺]_(o), each compartment was enveloped by afixed space in which potassium was allowed to accumulate. [K⁺]_(o)‘diffused’ either into the bathing solution or into astrocytes withK_(IR) kinetics. Since K_(IR) channel conductance is proportional to[K⁺]_(o) (Sakmann and Trube, 1984), K⁺ influx into ‘astrocytes’ wasdetermined by the local potassium gradient ([K⁺]_(o)−[K⁺]_(bath))modulated by K_(IR) conductance (log [K⁺]_(o); adapted from Ciani etal., 1978).

${\begin{matrix}{\Delta \left\lbrack K^{+} \right\rbrack}_{O} \\{\Delta \; t}\end{matrix} = {{\begin{matrix}{3\left( {I_{k} - I_{k}^{rest}} \right)} \\{VF}\end{matrix}\begin{pmatrix}1 & C\end{pmatrix}\begin{matrix}{\left\lbrack K^{+} \right\rbrack_{o} - \left\lbrack K^{+} \right\rbrack_{bath}} \\\tau_{ECS}\end{matrix}} - {C\frac{\left( {\left\lbrack K^{+} \right\rbrack_{o} - \left\lbrack K^{+} \right\rbrack_{bath}} \right)}{\tau_{astracytic}}{\log \left\lbrack K^{+} \right\rbrack}_{O}}}},$

where I_(k)—momentary K⁺ flux (nA/cm²), I_(k) ^(rest)—resting K⁺ flux(nA/cm²), τ_(ECS)—time constant for potassium diffusion into theextracellular space, τ_(astrocytic)—time constant for potassiumdiffusion into astrocytes, F—Faraday constant, C—ratio of astrocytic K⁺uptake relative to extracellular diffusion, and V—radius of envelopingextracellular space, set at 20 nm (Egelman and Montague, 1999;Savtchenko et al., 2000). The ionic flux equation describes first-orderpotassium clearance by both free diffusion and ‘astrocytic’ uptake (seeKager et al., 2000). Lateral diffusion of K⁺ ions was not taken intoaccount. To simulate a decrease in astrocytic potassium clearance,astrocytic was increased to mimic a reduction in astrocytic K_(IR)channels. “Resting” ion concentrations were set at (in [mM]): [Na⁺]_(o),145; [Na⁺]_(i), 12; [K⁺]_(o), 3.5; [K⁺]_(i), 140.

Statistical analysis. Data are expressed as means±SEM. Differencesbetween treated and control slices were determined by the Mann-Whitney Utest for two independent samples. Statistical tests were performed usingSPSS13.0 for Windows. The level of statistical significance was set atp<0.05, unless otherwise stated.

Results Astrocytic Transcriptional Changes Following BBB Opening orExposure to Albumin

To explore changes in astrocytic gene expression during epileptogenesis,gene-array data from DOC- and albumin-treated brains (n=3 from eachtreatment) were analyzed during the first 48 h after treatment and priorto the development of epileptiform activity (Ivens et al., 2007;Seiffert et al., 2004). When compared to sham-operated controls, the twotreatments, at each time point, resulted in similar changes inexpression of astrocytic-enriched genes with a correlation coefficientsbetween the different treatments (see Methods) of r²=0.69, 0.82, 0.85for 8, 24 and 48 h following treatment, respectively, p<0.0001, FIG. 10a). Unsupervised hierarchical cluster analysis revealed furthersimilarities between changes in transcripts levels in treated cortices(which cluster according to time after treatment) (FIG. 10 b) whiletranscripts changes in the contralateral, untreated, hemispheres arerelatively dissimilar and cluster together.

In a recent study, Cahoy and colleagues (2008) created a transcriptomedatabase reflecting cell type-specific, comprehensive mRNA expressionlevels in astrocytes, neurons and oligodendrocytes. We used these genelists to classify genes into “astrocytic” or “neuronal” categories. Whencompared with the “neuronal” category at all examined time points, the“astrocytic” category included a higher average number of genes thatunderwent a change in expression of more than ±150% (FIG. 10 c).Comparison between the results at 8 and 48 h after treatment showed anincrease in the average number of genes that reached 150% change in bothgroups (34 vs. 40 for astrocytes and 21 vs. 28 for neurons, 8 and 48 hafter treatment, respectively). The expression levels of genes reportedas over-expressed in reactive astrocytes (Ridet et al., 1997) was alsoexamined and found to show a large overlap with over-expressed genes 8,24 and 48 h following both treatments (FIG. 10 d). These results areconsistent with the hypothesis that an early and prominent change inastrocytic gene expression is an important early feature ofBBB-breakdown or albumin-induced epileptogenesis.

Altered Expression of Astrocytic Potassium and Glutamate RegulatingGenes

In their pioneering study, Kuffler and Potter (1964) established thatastrocytes are crucial for the control of the brain's extracellularenvironment. Specifically, these cells limit the accumulation of[K⁺]_(o) and glutamate (Oliet et al., 2001; Newman et al., 2004), thuspotentially contributing to the regulation of neuronal excitability. Thegene array results were searched for changes in the level of expressionof several potassium and glutamate homeostasis-related genes.Transcripts coding for the predominantly astrocytic (Kcnj10), but notneuronal (e.g. Kcnj2 or Kcncl, see Butt and Kalsi, 2006)inward-rectifying K⁺ channel (K_(IR)) were found to be down-regulated.In addition, the mRNA coding for the astrocytic glutamate transportersof the solute carrier family 1, subfamily A members SLC1A2 and SLC1A3(see Su et al., 2003; Chaudhry et al., 1995), but not for SLC1A4, wasdown-regulated. In contrast, SLC1A1 (preferentially expressed inneurons; see Rothstein et al., 1994) did not show significant changes inexpression levels. Glutaminase (Gls, Gls2) and glutamine synthetase(GS), both of which are predominantly expressed in astrocytes (Derouicheand Frotscher, 1991) and are responsible for regulating glutamatelevels, were also down-regulated (FIG. 11 a). Furthermore, the genearrays showed that at most time points there was a significantdown-regulation of gap junction proteins (Gja1, Gjb2, Gjb6) 24 hfollowing treatment, a finding that indicates reduced spatial bufferingcapacity (see Wallraff et al, 2006). Real-time RT-PCR confirmed the mainobservations obtained from the gene arrays, i.e., significantup-regulation of GFAP, and down-regulation of KCNJ10 (K_(IR) 4.1) aswell as KCNJ3, SLC1A2 and SLC1A3, Gja1, Gjb2 and Gjb6 at all time points(connexins 43, 26 and 30, respectively, FIG. 11 b). In contrast,glutamine synthetase did not show significant down-regulation (FIG. 11b).

The microarray results indicated a rapid and robust change in astrocyticgene expression in vivo following BBB breakdown or brain exposure toserum albumin. To further validate the specificity of the astrocyticresponse to albumin, cell cultures enriched with either astrocytes orneurons were exposed (see Methods) to albumin for 24 h. Significantly,the astrocytic cultures responded with significant down-regulation ofthe same transcripts found to respond in vivo to albumin (SLC1A3, GS,Gja1, Gjb2 and Gjb6, FIG. 11 c). No significant differences inexpression levels of the same transcripts were found in theneuronal-enriched culture (except for downregulation of GS andupregulation of Gjb6 mRNA levels, FIG. 11 d), confirming that thechanges observed in-vivo do indeed reflect an astrocytic response.

Epileptogenesis Involves Reduced Glutamate and Potassium Clearance

To confirm that the transcriptional changes induced by albumin wereassociated with altered cellular functions, the clearance ofextracellular glutamate and potassium in cortical slices 24 hoursfollowing albumin treatment in vivo was investigated. To measuresynaptic glutamate levels during neuronal activation, the slowlyinactivating (Lester et al., 1990) NMDA currents in cortical neuronswere recorded by using the whole-cell patch configuration (in thepresence of non-NMDA glutamate and GABA receptor blockers, see Methods).Cells were clamped at +40 mV to prevent a potential confounding effectof post-synaptic depolarization due to the accumulation of synaptic[K⁺]_(o). Mean single EPSC rise-time and amplitude were similar in bothcontrol and albumin-treated groups [14.5±0.5 vs. 13.2±0.7 ms and 505±100vs. 492±140 pA, for rise-time (not shown) and amplitude, respectively intreated vs. controls, FIG. 12 a, inset], suggesting that no changes inpost-synaptic NMDA receptor density or properties at this time point(data not shown). Synaptic glutamate elicited by 50 extracellularstimulations at 2, 5, 10, 20, 50 and 100 Hz before and after adding theastrocytic SLC1A2 specific inhibitor, DHK was measured. In neurons fromcontrol animals, DHK had no effect on single EPSCs or EPSCs elicited atlow stimulation frequencies (<20 Hz). In contrast, stimulationfrequencies>20 Hz resulted in increased NMDA currents (or reduceddepression when normalized to the first stimulus, FIG. 12 b-c, left),suggesting that astrocytic glutamate transporters efficiently reducesynaptic glutamate levels at high frequencies of neuronal activation.The same experiments were then repeated 24 h following corticalapplication of albumin (i.e. during epileptogenesis). In contrast to thecontrol experiments, DHK had no effect on EPSC amplitude in treatedslices (FIG. 13 b-c, right), supporting reduced expression of theastrocytic transporter SLC1A2. Repetitive stimulation, however, resultedin a stronger depression of EPSC amplitude in treated slices as comparedto controls.

To study K⁺ clearance from the extracellular space, K⁺ clearance wasrecorded from control and treated slices (24 h following treatment withalbumin) by using ISMEs. Slower decay kinetics of [K⁺]_(o) in responseto pressure application in BBB-treated animals has been reported (Ivenset al., 2007). Here, [K⁺]_(o) accumulation during neuronal activation atdifferent frequencies of stimulation was tested. In slices from controlanimals, the increase in [K⁺]_(o) was limited to 25% of baseline levels(<3.75 mM) at all stimulation frequencies with the employed stimulationintensities and number of stimuli. In contrast, in treated slices[K⁺]_(o) accumulation was significantly higher at frequencies≧10 Hz,reaching 6.7 mM (FIG. 12 d-e).

Modeling Reduced K⁺ Clearance Results in Frequency-DependentFacilitation of Excitatory Post-Synaptic Potentials

To elucidate the possible contribution of astrocytic dysfunction toneuronal excitability, a NEURON-based model of a post-synaptic neuronand an astrocyte was developed. To evaluate the role of increased[K⁺]_(o) accumulation and glutamate accumulation, changes to excitatorysynaptic currents in the post-synaptic neuron were examined (seeMethods). Excitatory synaptic input was simulated by simultaneousapplication of glutamate at all 8 distal dendritic processes (FIG. 13a). The reduction in K⁺ clearance was simulated by manipulating a[K^(+]) _(o)-regulated potassium removal mechanism (I_(KIR)), whilekeeping the diffusion component constant. In the absence of neuronalactivity, reducing K_(IR)-mediated potassium clearance had no effect onresting [K⁺]_(o) and thus had a negligible effect on the rising phaseand maximal amplitude of a single excitatory post-synaptic potential(EPSP) (FIG. 13 b). Reducing potassium buffering and consequentincreased K⁺ accumulation during repetitive stimulation resulted inenhanced EPSP duration due to slower repolarization (due to a reduceddriving force for K⁺ and a slight increase in NMDA-mediated current, seebelow and FIG. 14 b). During repetitive activation, the accumulation of[K⁺]_(o) near the dendritic compartment reached a maximum of 8.7 (and 16mM) for reduction to 50% (and 10%) of astrocytic [K⁺]_(o) bufferingcapacity, respectively (FIG. 13 c). [K⁺]_(o) accumulation duringrepetitive stimulation had a differential effect on AMPA- andNMDA-mediated currents: while the AMPA current showedfrequency-dependent depression due to receptor desensitization, the NMDAcomponent was strongly facilitated due to membrane depolarization (FIG.14 b). Reducing astrocytic potassium uptake from the extracellular spaceto 10% of control values resulted in an increase in total chargetransfer mediated by the NMDA component of 44, 344 and 84% at 10, 20 and100 Hz, respectively, while the AMPA charge transfer decreased by 5, 24,and 15%, respectively (FIG. 14 d). Overall, there was afrequency-dependent increase in EPSP amplitude (FIG. 14 e) associatedwith longer decay time (FIG. 13 f). Repeated simulations with no NMDAconductance (G_(NMDA)=0, with concomitant increased AMPA conductance, toachieve similar depolarization for a single stimulus) resulted in a muchsmaller facilitation (compare FIGS. 13 g and h).

Modeling Reduced Glutamate Clearance Results in Frequency-DependentDepression of Excitatory Post-Synaptic Potentials

The NEURON model and simulation paradigms described above were used totest the expected effect of reduced glutamate uptake. The reduction inglutamate uptake was simulated by slowing the transmitter's synapticdecay function. A twofold increase in the glutamate decay time constantresulted in a 48% increase in EPSP amplitude (from 25 to 37 mV) at asingle post-synaptic dendrite and a 60% increase in the amplitude of thesummated somatic EPSP (FIG. 14 a). While for a single stimulation bothAMPA and NMDA-components were increased, with repetitive activation, amarked decrease in the AMPA current (due to receptor desensitization,see Otis et al., 1996) and a strong facilitation of the NMDA current(due to post-synaptic depolarization, see Mayer et al., 1984 and FIG. 14b-c) were measured. Somatic EPSP facilitation (ratio of 5^(th) to 1^(st)EPSP amplitude, FIG. 15 c) was maximal at 100 Hz with our initialconditions for glutamate clearance. Inhibiting glutamate clearance didnot affect EPSP facilitation at low stimulation frequencies (<20 Hz) butreduced it at high stimulation frequencies (>80 Hz). The decreasedfacilitation was due to a reduced AMPA current through the desensitizedreceptors, thus keeping the membrane potential below the threshold forNMDA receptor activation. In simulations performed in the absence ofNMDA conductance, EPSP facilitation was reduced at most stimulationfrequencies, with only a small (<150%) residual facilitation measured athigh stimulation frequencies (>100 Hz, FIG. 14 d).

Modeling the Concerted Effect of Reduced Potassium and GlutamateClearance

The simulations showed that while synaptic glutamate levels mainlyaffected the 1^(st) EPSP in the train, an activity-dependent increase in[K⁺]₀ mainly enhanced EPSP facilitation in a frequency-dependent manner.Since the molecular data indicated a decrease in both potassium andglutamate buffering mechanisms, their joint effect on synaptictransmission was simulated. Decreasing the clearance of [K⁺]_(o) led tomaximal EPSP facilitation when stimulating at 20 Hz, while a concurrenttwo-fold reduction in glutamate uptake shifted the optimal frequency formaximal facilitation to 10 Hz (FIG. 15 a). Concurrent reductions inglutamate and [K⁺]_(o) clearance led to increases in the duration of the1^(st) EPSP, which in turn elicited increased and longer NMDA receptoractivation per stimulus. The longer EPSPs allowed for a larger chargetransfer with longer inter-stimulus intervals (i.e., reduced frequency,FIG. 15 b) thus lowering the optimal stimulation frequency. To assessthe sensitivity of the synaptic response during repetitive stimulation(at 20 Hz), several glutamate decay time constants and varying levels of[K⁺]_(o) uptake was used. We plotted the maximal EPSP amplitude as afunction of [K⁺]_(o); FIG. 15 c demonstrates that increasing synapticglutamate led to small increases in the maximal EPSP amplitudes for alllevels of [K⁺]_(o). However, synaptic facilitation was decreased withreduced glutamate uptake: thus, synaptic [K⁺]_(o) accumulation to 10 mMwas associated with 40% EPSP facilitation (upon the 5^(th) stimulation)under baseline glutamate clearance, but with only 22% facilitation whenglutamate decay time was doubled (FIG. 15 d).

Electrophysiological Evidence for Frequency-Dependent SynapticFacilitation During Epileptogenesis

The simulation data presented above predicted maximal EPSP facilitationat 20 Hz when [K⁺]_(o) clearance is reduced and decreased facilitation(at 50-100 Hz) when the only change induced is glutamate accumulation inthe synaptic cleft. Field potentials in response to stimulation atvarious frequencies in brain slices during “epileptogenesis” (exposureto albumin in sACSF) compared to controls (sACSF alone) were thereforemeasured. Comparison of the field potential amplitude and absoluteintegral during the first five stimuli revealed a significant reductionin both measures only under 100 Hz stimulation [amplitude: 1.13±0.12 vs.0.46±0.03 mV, 1.44±0.36 vs. 0.47±0.09 mV and area: 2.4±0.2 vs. 0.9±0.02V*s, 4.4±1.1 vs. 0.5±0.7 V*s, 1^(st) vs. 5^(th) stimulus, control (n=5)and treated (n=4), respectively, p<0.05]. Comparing field potentialduration (measured at ⅓ maximal amplitude) for the 1^(st) vs. the 5^(th)stimulus among different frequencies did not reveal any changes incontrol slices. In contrast, in treated slices the field potential wassignificantly prolonged at 10 and 20 Hz (10 Hz: 7.5±0.4 vs. 9.4±5.5 ms,6.5±0.7 vs. 13.1±2.6 ms, 20 Hz: 6.0±0.9 vs. 6.8±0.8 ms, 6.6±0.8 vs.12.9±2.9 ms for 1^(st) vs. 5^(th) stimulus in control and treated,respectively, p<0.05, FIG. 16 c). Interestingly, in the “treated” group,stimulation-induced frequency-dependent, long-lasting epileptiformdischarges occurred most reliably during 10-Hz stimulation (4 of 4slices, n=3 animals), and sometimes at 20 Hz (3 of 4 slices) and 5 Hz (2of 4 slices), but never at higher frequencies (FIG. 16 d). Epileptiformdischarges were observed in one control slice without any apparentfrequency dependence (5 to 50 Hz, 1 of 5 slices, n=3 animals, FIG. 16d).

Taken together, these experiments show that exposure to albumin in-vitroinduces changes in neuronal excitability and that evoked networkactivity facilitates, and often turns into, robust epileptiformdischarges upon repetitive stimulation. 10-20 Hz was found to be themost reliable frequency, as was also predicted by the K⁺ recording data(FIG. 12 d) and by the model shown above in the case of reduction in[K⁺]_(o) clearance with or without glutamate accumulation (see FIG. 13g).

FIGS. 10-16

FIG. 10. Transcriptional changes in astrocytes following exposure toalbumin or BBB disruption. (a) Sham-normalized expression levels of mRNAfor genes preferentially expressed in astrocytes at 8, 24, and 48 hfollowing treatment with the BBB disrupting agent DOC (D) or albumin(A). (b) Hierarchical cluster analysis comparing astrocytic geneexpression for DOC-treated and albumin-treated cortices at 8, 24, and 48h following treatment, and the contralateral, non-treated hemisphere(Ctrl Hemi.). (c) Average number of gene transcripts up- ordown-regulated by more than 150% grouped by cell-type across all timepoints. (d) Sham normalized mRNA expression levels of genes coding forknown astrocytic activation markers.

FIG. 11. Alterations in astrocytic potassium and glutamate regulatinggenes. (a) Sham-normalized mRNA expression levels for genes associatedwith K+ and glutamate homeostasis at 8, 24, and 48 h following in-vivotreatment with DOC (D) and albumin (A). (b) Sham-normalized mRNAexpression levels for selected transcripts (see Results) obtained byreal-time RT-PCR 24 h following DOC (grey bars) or albumin (black bars)treatments. (c) Astrocyte enriched cell cultures immunostained for GFAP(red) or NeuN (green). Nuclei visualized with DAPI staining (blue). Thegraph shows mRNA expression levels in albumin-exposed cultures comparedto controls. (d) same as c, for neuron enriched cultures. Abbr.:SLC1a2-GLT-1, SLC1a3-GLAST, Gja1, Gjn2, Gjb6, -connexins 43, 26, 30respectively, Kcnj3-KIR3.1

FIG. 12. Electrophysiological evidence for reduced glutamate andpotassium buffering during epileptogenesis. (a) Single NMDA-mediatedEPSCs in control slices and 24 h following albumin treatment in vivo inACSF (gray) and 10 min following DHK (black). Inset shows mean EPSCamplitude in ACSF. (b) NMDA-mediated EPSCs during train stimulation at50 Hz in control animals and treated animals. (c) Mean evokedNMDA-mediated EPSC at different stimulation frequencies. (d) [K+]olevels in control and treated slices during 20 Hz stimulation (e) MeanK+]o levels during extracellular stimulation at 2-100 Hz (right). #,p<0.03, *, p<0.001 (n=6 albumin-treated cells, 5 animals, n=9 controlcells, 7 animals).

FIG. 13. Application of NEURON-based model to determine the effects of[K+]o accumulation. (a) Schematic diagram of the modeled layer 2/3pyramidal neuron containing 74 compartments with 8 synapses (with AMPAand NMDA currents), one at each distal dendrite. (b) Increasingglutamate levels at each of the 8 synapses (top trace representingkinetics of synaptic glutamate level) elicits AMPA-mediated (middletrace) and NMDA-mediated (bottom trace) currents under controlconditions (black) and under reduced astrocytic potassium clearance(under a 10-fold decrease of astrocytic K+ clearance, blue trace). (c)Maximal K+ concentrations recorded in the vicinity of a distal dendriticcompartment during repetitive stimulation as a function of [K+]oclearance and stimulation frequency. (d) Percent change of total chargetransfer by NMDA (black) and AMPA (blue) channels during stimulation at20 and 100 Hz. (e). Somatic EPSPs under control conditions (black) andunder reduced astrocytic potassium clearance (10-fold reduction ofcontrol levels, blue trace). (f) 5th EPSP (elicited by stimulation at 4,20, and 100 Hz) decay time constant at different levels of astrocyticpotassium clearance rates. (g) Ratio of 5th to 1st EPSP amplitude(P5/P1) at stimulation frequencies of 4-500 Hz at different levels ofastrocytic potassium clearance rates. (h) Same as in (g) with GNMDA=0.

FIG. 14. Application of NEURON-based model to determine the effects ofglutamate accumulation. (a) Somatic EPSP amplitudes for differentglutamate time constants. (b) Simultaneous glutamate “application”(kinetics represented in the upper trace) at each of the 8 synapseselicits AMPA-mediated (middle) and NMDA-mediated (bottom) currents undercontrol conditions (black) and a twofold increase in glutamate decaytime constant (blue). (c) Ratio of 5th to 1st EPSP amplitude (P5/P1) atdifferent stimulation frequencies and varying glutamate decay timeconstants (values related to control). (d) Same as in (c) with GNMDA=0.

FIG. 15. Modeling the concerted effect of reduced potassium andglutamate clearance. (a) EPSP facilitation (relative to maximal value)for a 10-fold decrease in [K+]o clearance (black), a twofold slowing ofglutamate decay time constant (red) and for down regulation of bothuptake mechanisms (blue) as a function of stimulation frequency. (b)EPSP traces for 10- and 20-Hz trains under a 10-fold decrease inastrocytic K⁺ clearance (gray and blue traces, respectively) and withboth uptake mechanisms down regulated (at 10 Hz, black). Dashed linemarks resting potential. (c) Maximal EPSP amplitude elicited by a trainof five stimuli as a function of maximal [K+]o for different glutamateuptake decay time constants (for 1.2, 2.2, 3.2 and 3.5 ms). (d) EPSPsfacilitation [ratio of 5th to 1st EPSP amplitude (P5/P1)] for 20 Hzstimulation for different glutamate decay time constants (as in c).

FIG. 16. Recording in vitro shows frequency-dependent increased neuronalexcitability and hyper-synchronous network activity duringalbumin-mediated epileptogenesis. (a, b) Neocortical field potentialrecordings of brain slices during stimulation trains of 50 pulses at 2,10 and 100 Hz. Field responses were facilitated in the albumin-treatedslices, observed as increased duration of the population spikes (seeinset in a, b). (c) Comparison of the average field potential duration(at ⅓ maximal amplitude) for the 5th to the 1st evoked response revealsmaximal facilitation at 10 Hz. (d) Percentage of slices showingprolonged, paroxysmal discharges.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of treating a neurological disorder inan individual, the method comprising administering to the individual aneffective amount of a transforming growth factor-beta (TGF-β) pathwayblocker.
 2. The method of claim 1, wherein the TGF-β pathway blockerspecifically inhibits kinase activity of TGF-βI.
 3. The method of claim1, wherein the TGF-β pathway blocker is an aldosterone inhibitor.
 4. Themethod of claim 3, wherein the aldosterone inhibitor is7α,11α,17α)-pregn-4-ene-7,21-dicarboxylicacid,9,11-epoxy-17-hydroxy-3-oxo-γ-lactone, methyl ester or7α-acetylthio-3-oxo-17α-pregn-4-ene-21,17-carbolactone.
 5. The method ofclaim 1, wherein the TGF-β pathway blocker is an angiotensin II receptorinhibitor.
 6. The method of claim 5, wherein the angiotensin II receptorinhibitor is2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-methanol,N-[p-(o-1H-tetrazol-5-yl-phenyl)benzyl]-N-valeryl-L-valine,2-n-butyl-4-spirocyclopentane-1-((2′-tetrazol-5-yl)biphenyl-4-yl)-2-imidazolin-5-one,1-(cyclohexyloxycarbonyloxy)ethyl-2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazole-7-carboxylate,4′-[(1,4′-dimethyl-2′-propyl[2,6-bi-1H-benzimidazol]-1-yl]methyl]-[1,1′-biphenyl]-2-carboxylicacid,5,8-dihydro-2,4-dimethyl-8-[p-(o-1H-tetrazol-5-ylphenyl]pyrido[2,3-d]pyrimidin-7(6H)-one,or4-({2-butyl-5-[2-carboxy-2-(thiophen-2-ylmethyl)eth-1-en-1-yl]-1H-imidazol-1-yl}methyl)benzoicacid, or a pharmaceutically acceptable salt of any of the foregoing. 7.The method of claim 5, wherein the angiotensin II receptor inhibitor is2-butyl-4-chloro-1-[p-(o-1H-tetrazol-5-ylphenyl)benzyl]imidazole-5-methanol,or a pharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein the TGF-β pathway blocker is an angiotensin converting enzyme(ACE) inhibitor.
 9. The method of claim 1, wherein the TGF-β pathwayblocker is a renin inhibitor.
 10. The method of claim 1, wherein theTGF-β pathway blocker is a proteoglycan selected from decorin, biglycan,fibromodulin, lumican, betaglycan and endoglin.
 11. The method of claim1, wherein the TGF-β pathway blocker is an agent that reduces the leveland/or activity of Smad1.
 12. The method of claim 11, wherein the agentthat reduces the level and/or activity of Smad1 is3-(6-Methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole,2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine, or4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide13. The method of claim 1, wherein the TGF-β pathway blocker reduces thelevel and/or activity of NFκB.
 14. The method of claim 1, wherein theTGF-β pathway blocker is a mitogen activated protein kinase inhibitor.15. The method of claim 1, wherein the TGF-β pathway blocker is aninhibitory nucleic acid that reduces the level of a TGF-β pathwayelement.
 16. The method of claim 1, wherein the neurological disorder isepilepsy.
 17. The method of claim 1, wherein the neurological disorderis stroke.
 18. The method of claim 1, wherein the neurological disorderresults from traumatic head injury.
 19. The method of claim 1, whereinthe neurological disorder is a neurodegenerative disorder.
 20. Themethod of claim 1, wherein a single TGF-β pathway blocker isadministered in monotherapy.
 21. The method of claim 1, wherein a singleTGF-β pathway blocker is administered in combination therapy with atleast one additional therapeutic agent other than a TGF-β pathwayblocker.
 22. The method of claim 1, wherein the TGF-β pathway blocker isadministered via injection.
 23. The method of claim 1, wherein the TGF-βpathway blocker is administered orally.
 24. The method of claim 1,wherein the TGF-β pathway blocker is administered intracranially. 25.The method of claim 1, wherein the TGF-β pathway blocker is administeredtogether with an agent that facilitates crossing the blood-brainbarrier.